Soluble mammalian adenylyl cyclase and uses therefor

ABSTRACT

A soluble form of adenylyl cyclase and methods of its use in screening for stimulators and inhibitors of adenylyl cyclase activity are disclosed. In one embodiment, a chimera of type I and type II adenylyl cyclases is provided. This chimera lacks transmembrane domains characteristic of adenylyl cyclases, rendering the recombinant product soluble, while retaining adenylyl cyclase function.

The government may own certain rights in the present invention pursuant to grant number GM34497 from the National Institutes of Health.

The present application claims benefit of the priority accorded to provisional application Ser. No. 60/005,498 filed on Oct. 4, 1995. The entire text of the aforementioned disclosure is specifically incorporated by reference herein without disclaimer.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of molecular pharmacology. More specifically, the invention involves the molecular biology of the adenylyl cyclase pathway and, in particular, the structure and function of the enzyme adenylyl cyclase.

2. Description of the Related Art

Cyclic AMP regulates intracellular reactions in all nucleated animal cells studied to date. The system by which cyclic AMP is produced is the adenylyl cyclase system, which comprises G-protein coupled receptors, G-proteins and the catalytic, membrane-bound enzyme known as adenylyl cyclase. Adenylyl cyclases have molecular weights of about 120 kD and are stimulated directly by the diterpene forskolin.

The structures of G protein-regulated adenylyl cyclases are complex, consisting of two intensely hydrophobic domains (M₁ and M₂ with each hypothesized to contain six transmembrane helices) and two approximately 40 kD cytosolic domains (C₁ and C₂). C₁ and C₂ contain sequences (C_(1a) and C_(2a)) that are similar to each other, to corresponding regions of related adenylyl cyclases and to the catalytic domains of the related membrane-bound, soluble guanylyl cyclases (Tang and Gilman, 1992). Analysis of a series of truncation and alanine-scanning mutants of mammalian adenylyl cyclases indicated that both C_(1a) and C_(2a) (but not C_(1b) and C_(2b)) are necessary for catalytic activity (Tang et al., 1992).

Unfortunately, membrane-bound adenylyl cyclases are found in small amounts and the enzymes are both labile and difficult to manipulate in detergent-containing solutions. As a result, biochemical studies on the mechanism of regulation of adenylyl cyclases have been relatively unrewarding. Improved methods for isolation of this enzyme, relying on a forskolin affinity matrix, have permitted some purification. Recombinant expression has proved difficult as well, as the significant transmembrane regions of adenylyl cyclases create significant technical problems, especially in prokaryotic expression systems. Thus, there remains a need to develop improved reagents and assays that can be used to further characterize adenylyl cyclase and to screen for compounds that stimulate and inhibit adenylyl cyclase activity.

SUMMARY OF THE INVENTION

In light of the limitations described above, a goal of the present invention is to provide soluble forms of adenylyl cyclase that display normal regulatory function, and methods of production thereof. In addition, it is a goal to provide assays for the screening of inhibitors of adenylyl cyclase activity.

Thus, it is a goal of the present invention to provide adenylyl cyclase compositions that will be suitable for use in exploring the function of this enzyme. In addition, it is a goal to provide methods of synthesizing these compositions. Finally, it is a goal to provide methods of identifying inhibitors of adenylyl cyclase.

In fulfilling these goals, there is provided a soluble polypeptide composition having adenylyl cyclase activity. The polypeptide composition generally comprises one or more polypeptides that lack transmembrane regions.

In one embodiment, the polypeptide composition comprises a chimera of adenylyl cyclase C₁ and C₂ domains linked covalently. For example, the chimera may consist essentially of adenylyl cyclase type I-C₁ and type II-C₂ domains, more specifically, wherein the type I-C₁ domain is a C_(1a) domain sequence from SEQ ID NO:2 and the type II-C₂ domain is a C_(2a) domain sequence from SEQ ID NO:4. In another example, the chimera consists essentially of adenylyl cyclase type V-C₁ and type II-C₂ domains. The domains may be joined by a linker peptide, for example, a linker peptide having a sequence selected from the group consisting of AAAGGM (SEQ ID NO:19), AAAGGMPPAAAGGM (SEQ ID NO:20) and AAAGGM(PPAAAGGM)₂ (SEQ ID NO:21).

In an alternative embodiment, the polypeptide composition according to claim 2, wherein the polypeptide composition forms a complex comprising two distinct polypeptides, one of which is an adenylyl cyclase C₁ domain and one of which is an adenylyl cyclase C₂ domain. For example, the complex has a C₁ domain that is a type I C₁ domain and a C₂ domain that is a type II C₂ domain, more specifically, the type I-C₁ domain has a C_(1a) domain sequence from SEQ ID NO:2 and the type II-C₂ domain has a C_(2a) domain sequence from SEQ ID NO:4. Alternatively, the C₁ domain is a type V C₁ domain the C₂ domain is a type II C₂ domain.

In another embodiment of the present invention, there is provided a polynucleotide encoding a soluble polypeptide having adenylyl cyclase activity. Generally, the polynucleotide does not encode transmembrane regions. In one embodiment, the polynucleotide encodes a chimera of adenylyl cyclase C₁ and C₂ domains, for example, as in SEQ ID NO:17. Alternatively, the polynucleotide encode an adenylyl cyclase C₁ or C₂ domain. Expression vectors, wherein these polynucleotides are operably linked to a promoter, and host cells comprising such expression vectors, also are contemplated. The host cells may be bacterial in origin or they may be mammalian cells, especially insect cells that are capable of supporting baculovirus replication. In a specific embodiment, the host cell further comprises an expression vector comprising a polynucleotide encoding the alpha subunit of G protein, operably linked to a promoter active in the host cell.

In yet another embodiment, there is provided a method for determining the effects of an agent on adenylyl cyclase activity comprising the steps of (i) providing a soluble polypeptide composition having adenylyl cyclase activity; (ii) contacting the polypeptide composition with the agent; and (iii) measuring the adenylyl cyclase activity of the polypeptide composition. The polypeptide composition generally will lack a transmembrane region. In one embodiment, the polypeptide composition is a chimera of adenylyl cyclase type C₁ and C₂ domains. In another embodiment, the composition forms a complex comprising two distinct polypeptides, one of which is an adenylyl cyclase C₁ domain and one of which is an adenylyl cyclase C₂ domain.

As part of this method, the providing may comprise transforming a host cell with an expression vector comprising a polynucleotide encoding the chimera, operably linked to a promoter active in the host cell. The method also may further comprise transforming the host cell with an expression vector comprising a polynucleotide encoding the alpha subunit of G protein, operably linked to a promoter active in the host cell. The growth of the host cell may be conditional on adenylyl cyclase activity and the measuring comprises assessing the growth of the host cell, for example, growth on minimal agar. Alternatively, the method may function such that the maltose utilization of the host cell is conditional on the absence of adenylyl cyclase activity and the measuring comprises assessing the maltose utilization of the host cell, wherein the transformed host cell is a bacterial cell and growth is on McConkey agar.

In an alternatively embodiment, the providing step may comprise transforming a host cell with two expression vectors, one of the expression vectors comprising a polynucleotide encoding an adenylyl cyclase C₁ domain, operably linked to a promoter active in the host cell, and the other expression vector comprising a polynucleotide encoding an adenylyl cyclase C₂ domain, operably linked to a promoter active in the host cell. Finally, the providing step may comprise (a) transforming a first host cell with a first expression vector comprising a polynucleotide encoding an adenylyl cyclase C₁ domain, operably linked to a promoter active in the host cell, (b) transforming a second host cell with a second expression vector comprising a polynucleotide encoding an adenylyl cyclase C₂ domain, operably linked to a promoter active in the host cell, (c) isolating the C₁ and C₂ domains, and (d) admixing the isolated C₁ and C₂ domains under conditions permitting formation of the complex.

In still yet another embodiment, there is provided a method of producing a soluble adenylyl cyclase complex comprising the steps of (a) transforming a first host cell with a first expression vector comprising a polynucleotide encoding an adenylyl cyclase C₁ domain, operably linked to a promoter active in the host cell, (b) transforming a second host cell with a second expression vector comprising a polynucleotide encoding an adenylyl cyclase C₂ domain, operably linked to a promoter active in the host cell, (c) isolating the C₁ and C₂ domains, and (d) admixing the isolated C₁ and C₂ domains under conditions permitting formation of the complex. The complex may have a C₁ domain that is a type I C₁ domain and a C₂ domain that is a type II C₂ domain. Alternatively, the complex may have a C₁ domain that is a type V C₁ domain and a C₂ domain that is a type II C₂ domain.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein:

FIG. 1A and FIG. 1B--Chimeric adenlyl cyclase. A model of mammalian adenylyl cyclase (FIG. 1A) and the various chimeras between type I and type II adenylyl cyclases (FIG. 1B) are shown. Also illustrated are the linker sequences (SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21. IC_(1a) includes residues 271-484 of type I adenylyl cyclase; IIC_(2a) includes residues 821-1090 of type II adenylyl cyclase. No sequences from any of the putative transmembrane spans of either enzyme are included.

FIG. 2--Enzyme activity of chimeras. Adenylyl cyclase activities of supernatant fractions of lysates (20 μg) from E. coli containing the indicated plasmids were assayed with 10 mM MgCl₂ at 30° C. for 30 min. Assays also contained 200 nM GTP-γ-S-G_(s)α), 100 μM forskolin (Fsk), or 100 μM CaCl₂ plus 2 μM calmodulin, as indicated.

FIG. 3--Enzymatic activity of IC₁ IIC₂ -L₃. Activation of soluble adenylyl cyclase by forskolin, but not by 1,9-dideoxyforskolin.

FIG. 4--Activation by GTP-γ-S-G_(s)α. Activation of soluble adenylyl cyclase by GTP-γ-S-G.sub.α.

FIG. 5--Synergistic activation by GTP-γ-S-G_(s)α and forskolin. The concentration of GTP-γ-S-G_(s)α was 200 nM (same in FIG. 6 and FIG. 7). Sum [Fsk+G_(s)α ] is the sum of adenylyl cyclase activities observed in the presence of forskolin or GTP-γ-S-G_(s)α alone. Fsk+G_(s)α is adenylyl cyclase activity observed in the presence of both GTP-γ-S-G_(s)α and forskolin (same in FIG. 6 and FIG. 7).

FIG. 6--Determination of K_(m),ATP. The Michaelis constant for ATP forskolin or GTP-γ-S-G_(s)α. The concentration of forskolin was 10 μM (same in FIG. 7).

FIG. 7A and FIG. 7B--Effect of 2'-deoxy-3'-AMP. The adenylyl cyclase activity of a supernatant fraction (20 μg) from E. coli expressing IC₁ IIC₂ -L₃ was assayed at 30° C. for 30 min. In FIG. 7B, adenylyl cyclase was activated with forskolin. In FIG. 7A, adenylyl cyclase was assayed with forskolin (∘), GTPγS-G_(s)α (), or forskolin plus GTPγS-G_(s)α (□); control activities for these conditions were 270, 95 and 970 pmol/min/mg, respectively. Data shown are representative of at least two studies.

FIG. 8--Superdex 75 gel filtration chromatography of an extract containing IC₁ IIC₂ -L₃. Molecular weight markers are thyroglobulin (670 kD), gamma globulin (158 kD), chicken ovalbumin (44 kD) and horse myoglobin (17 kD). Data shown are representative of two studies.

FIG. 9. Simple mixture of the C_(1A) and C₂ domains of adenlyl cyclase reconstitutes adenylyl cyclase activity. Bacterial lysates containing either the IC₁ or the IIC₂ fragments of adenylyl cyclase were assayed as described by themselves (20 μg) or after mixture (10 μg of each) with either no activator, 50 μM FSK, or 50 μM FSK plus 200 nM GTP[γS]-G_(s)α. Activities shown as 0 represent less than 1 pmol/min, the limit of detection.

FIG. 10A and FIG. 10B. Definition of a reconstitutive assay for purification of IC₁ and IIC₂. (FIG. 12A) Increasing amounts of lysate containing IC₁ were mixed with 1.5 μg of the Ni²⁺ -NTA column eluate containing IIC₂ and assayed with 50 μM FSK. (FIG. 10B) Increasing amounts of a lysate containing IIC₂ were mixed with 1 μg of the Ni²⁺ -NTA column eluate containing IC₁ and assayed with 50 μM FSK.

FIG. 11A and FIG. 11B. Purification of IIC₂. (FIG. 11A) Mono Q column chromatography. Fractions were 6 ml. (FIG. 11B) Gel filtration over Superdex 200. Fractions were 0.5 ml. The positions of molecular weight markers are shown.

FIG. 12A and FIG. 12B. Purification of IC₁. (FIG. 12A) Phenyl-Sepharose column chromatography. Fractions were 2 ml. The first peak of activity was pooled for further purification. (FIG. 12B) Gel filtration over Superdex 200. Fractions were 0.5 ml. The positions of molecular weight markers are shown.

FIG. 13A and FIG. 13B. Interactions of IC₁ with IIC₂. (FIG. 13A) The indicated concentrations of IIC₂ were assayed in the presence of 0.1 μM IC₂ for 30 min. In the absence of activators. (FIG. 13B) The indicated concentrations of IIC₂ were assayed in the presence of 8 nM IC₁ for 10 min. With either 50 μM FSK () or 50 μM FSK plus 10 μM GTP[γS]-G_(s)α (). Activities are expressed per mg of IC₁.

FIG. 14A and FIG. 14B. Interactions between FSK and GTP[γS]-G_(s)α. (FIG. 14A) Fragments IIC₂ (6.6 μM) and IC₁ (8 nM) were mixed and assayed in the presence of the indicated concentrations of FSK, with (▪) or without () 0.5 μM GTP[γS]-G_(s)α (FIG. 14B) Fragments IIC₂ (6.6 μM) and IC₁ (8 nM) were mixed and assayed in the presence of the indicated concentrations of GTP[γS]-G_(s)α, with (▪) or without () 50 μM FSK.

FIG. 15. A simple model for the interactions of IC₁, IIC₂, G_(s)α, and FSK. The two domains of adenylyl cyclase are designated C_(x) and C_(y) where their identities are unknown; they are designated C₁ and C₂ when associated.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

One of the primary reasons that research on adenylyl cyclase has been limited is that it has been difficult to purify significant quantities of the active enzyme. Even when improved methods permitted isolation, the ability of the purified enzyme has further hampered efforts. An alternative--recombinant production--also has proved problematic, largely because of the two hydrophobic transmembrane regions found in adenylyl cyclases. It has proved difficult to synthesize active enzymes containing significant transmembrane regions because of the loss of higher order structure in the absence of membranes. The present invention has overcome this problem by providing adenylyl cyclase compositions lacking transmembrane regions but retaining activity and characteristic regulatory features. The following describes the present invention in detail.

1. Adenyl Cyclase and Nucleic Acids Coding Therefor

Adenylyl cyclases are enzymes of approximately 120 kD (1064-1248 amino acids). There currently are eight different isoforms known, designated I-VIII. The typical structure of adenylyl cyclase begins with a short cytoplasmic amino terminus followed by a first hydrophobic region comprising six transmembrane spans (M₁). This is followed by a first cytoplasmic domain of about 40 kD (C₁). A second six-span transmembrane region (M₂) and a second cytoplasmic domain (C₂) follow. While there is no evidence that adenylyl cyclases are membrane channels or transporters, their structure is reminiscent of molecules having these functions.

The overall amino acid sequence similarity among the different adenylyl cyclases is roughly 50%. Within each of the two cytoplasmic regions, there are more highly conserved regions, designated C_(1a) and C_(2a). These domains show considerable homology between isoforms and across species of adenylyl cyclases. In addition, C_(1a) and C_(2a) are structurally related to the catalytic domains of guanylyl cyclases. Interestingly, C_(1a) and C_(2a) show similarity to each other as well.

Because of the difficulties associated with recombinant expression of polypeptides containing membrane-spanning regions, it is desirable to identify functional catalytic subunits within adenylyl cyclases. Attempts to detect adenylyl cyclase activity following expression of the C_(1a) or C_(2a) regions individually have not been successful, however. Similarly, individual expression of C₁, C₂, M₁ C₁ or M₂ C₂ has not been accompanied by detectable cyclase activity.

When M₁ C₁ and M₂ C₂ are coexpressed in the same cell, however, cyclase activity is observed. Thus, at a minimum, function of adenylyl cyclase apparently requires some level of interaction between the C₁ and C₂ domains. Whether or not the transmembrane regions were necessary for this function remained unknown prior to the present invention. As shown herein, however, chimeras and non-covalent complexes of the C_(1a) and C_(2a) domains of adenylyl cyclases types I and II, respectively, which completely lack the membrane-spanning regions, also exhibit regulated adenylyl cyclase activity. This also suggests that C₁ and C₂ interaction is important and, further demonstrates that the transmembrane regions are not required for enzymatic function. It is further demonstrated that mixtures of C1 and C2 domains adenylyl cyclases of types I, II and V, likewise completely lacking membrane spanning regions exhibit regulatable adenylyl cyclase activity.

Some other structural constraints on adenylyl cyclase function may also exist. Studies indicate that one particular recombinant enzyme consisting of the C_(1a) and C_(2a) regions of type I adenylyl cyclase, made as described herein, may not be active. Alternatively, this lack of activity may be a function of the kind of linkage used to connect the two domains. A similar type I/type II chimera is functional, however. Given the instant disclosure, it would be a simple matter to synthesize any of the 64 possible combinations of C_(1a) and C_(2a) regions to determine which provide functional enzymes (Table 1) using the techniques described in the Examples.

                                      TABLE 1                                      __________________________________________________________________________     The possible combinations of C1a and C2a                                             C1a Type                                                                 I         II  III IV  V   VI  VII VIII                                         __________________________________________________________________________     C2a                                                                               I  I/I II/I                                                                               III/I                                                                              IV/I                                                                               V/I VI/I                                                                               VII/I                                                                              VIII/I                                         Type II I/II II/II III/II IV/II V/II VI/II VII/II VIII/II                       III I/III II/III III/III IV/III V/III VI/III VII/III VIII/III                  IV I/IV II/IV III/IV IV/IV V/IV VI/VI VII/IV VIII/IV                           V I/V II/V III/V IV/V V/V VI/V VII/V VIII/V                                    VI I/VI II/VI III/VI IV/VI V/VI VI/VI VII/VI VIII/VI                           VII II/VII II/VII III/VII IV/VII V/VII VI/VII VII/VII VIII/VII                                                  VIII I/VIII II/VIII III/VIII IV/VIII                                         V/VIII VI/VIII VII/VIII VIII/VIII            __________________________________________________________________________

The nucleic acid and amino acid sequences for the various adenylyl cyclases are known to those of skill in the art, are described in numerous published articles, and are available from publicly accessible databases. By way of example only, Krupinski et al. (1989) describe the nucleic acid and amino acid sequences for type I adenylyl cyclase; Feinstein et al. (1991) describe type II adenylyl cyclase sequences; Bakalyar & Reed (1990) describe type III adenylyl cyclase sequences; Gao & Gilman (1991) describe type IV adenylyl cyclase sequences; Premont et al. (1992) describe type V and VI adenylyl cyclase sequences; Krupinski et al. (1992) and Katsushika et al. (1992) particularly describe VI adenylyl cyclase sequences; Watson et al. (1994) describe type VII adenylyl cyclase sequences; and Cali et al. (1994) describe type VIII adenylyl cyclase sequences. The foregoing articles and others concerning adenylyl cyclase genes and cDNAs, such as Yoshimura & Cooper (1992) and Wallach et al. (1994), are each incorporated herein by reference.

In addition to the foregoing articles, various exemplary mammalian adenylyl cyclase nucleic acid and amino acid sequences are provided in the present sequence listing. The nucleic acid and amino acid sequence of type I are exemplified by SEQ ID NO:1 and SEQ ID NO:2, respectively. Similarly, SEQ ID NO:3 and SEQ ID NO:4 represent type II adenylyl cyclase; SEQ ID NO:5 and SEQ ID NO:6 represent type III; SEQ ID NO:7 and SEQ ID NO:8 represent type IV; SEQ ID NO:9 and SEQ ID NO:10 represent type V; SEQ ID NO:11 and SEQ ID NO:12 represent type VI; SEQ ID NO:13 and SEQ ID NO:14 represent type VII; and SEQ ID NO:15 and SEQ ID NO:16 represent type VIII adenylyl cyclase.

The nucleic acid and amino acid sequences for the exemplified chimera are provided in SEQ ID NO:17 and SEQ ID NO:18, respectively. Various other chimeric constructs are readily preparable using the sequence information of the present disclosure and standard molecular biological linker technology, e.g., using the peptide linker sequences in combination with the codon information in Table 4.

One of the important benefits deriving from the deletion of the transmembrane regions is the production of an adenylyl cyclase that is soluble. "Soluble" is defined here in as capable of dissolving in a aqueous environment in the absence of detergent.

It will be understood that the soluble adenylyl cyclase compositions provided by the invention represents a significant advantage over the prior art in that the previous methods required either preparation of membrane fractions (e.g., Feinstein et al., 1991; Katsushika et al., 1992; Cali et al., 1994) or solubilization in detergent (see e.g., Tang et al., 1991). Even expression of the protein in a membrane environment was not always successful. For example, Gao and Gilman (1991) reported that expression of type IV adenylyl cyclase in Sf9 cell membranes was associated with a considerable amount of denaturation and/or aggregation.

A detergent commonly used prior to the present invention is Lubrol PX. However, even using detergent solubilization, the prior art methods were not always successful in extracting or isolating recombinant adenylyl cyclase. For example Tang et al. (1991) reported only a 50-60% efficiency of solubilization for recombinant adenylyl cyclase (Table I of Tang et al., 1991).

Other detergents that have been used previously in attempts to solubilize and purify adenylyl cyclase include cholate, digitonin, CHAPS, octylglucoside and dodecylmaltoside in the presence of glycerol and NaCl (Taussig et al., 1994). Therefore, this invention may be characterized as an adenylyl cyclase preparation that is soluble in an aqueous environment in the absence of a significant amount of a detergent such as Lubrol PX, cholate, digitonin, CHAPS, octylglucoside or dodecylmaltoside in the presence of glycerol and NaCl.

Also, in the prior art methods, in further purification from the initial detergent solubilization step, recovery of the adenylyl cyclase protein was reported to be only about 10% of the total protein (Tang et al., 1991). Furthermore, after purification, the adenylyl cyclase protein of the prior art did not exhibit the expected properties, such as being inhibited by βγ, even though the impure protein preparation did exhibit such properties. By providing soluble adenylyl cyclase that displays normal properties and regulatory functions, the present invention marks a breakthrough in this area.

It is contemplated that the precise form of the chimeric or complexed adenylyl cyclase compositions may vary without adversely affecting its function. For example, there is no particular constraint on the precise size of the catalytic domains and their potential fusion sites so long as essential sequences are included. Similarly, in the chimeric compositions the mode of fusion (direct versus spacer linked) may differ from construct to construct.

The domains of the soluble adenylyl cyclase chimeric compositions will likely most often be joined by a spacer peptide, generally of a flexible nature, although other chemical linkages are not excluded. Currently, it is contemplated that the most useful linker sequences will generally be peptides of between about 6 and about 40 amino acids in length, or so, with linkers of between about 6 and about 25 amino acids in length being more preferred. These linkers are produced by using synthetic, linker-encoding oligonucleotides to couple the C_(1a) and C_(2a) coding regions.

Peptide linkers with a degree of flexibility will generally be preferred. The linking peptides may have virtually any amino acid sequence, bearing in mind that the preferred linkers will have a sequence that results in a generally flexible peptide. The use of small amino acids, such as glycine and alanine, will likely be of use in creating a flexible peptide. The creation of such sequences will be routine to those of skill in the art.

A variety of different linkers are commercially available and are considered suitable for use according to the present invention. Amino acid sequences rich in alanine and proline residues are known to impart flexibility to multi-domain protein structures. For example, such sequences are to be found linking the domains of the so-called E2 components of the 2-oxo acid dehydrogenase complexes, such as pyruvate dehydrogenase complex and 2-oxo glutarate dehydrogenase complex (Perham et al., 1981; Perham & Roberts, 1981; Texter et al., 1988; Miles et al., 1988; Radford et al., 1987, 1989). Alanine-proline rich regions are also found in myosin light chains (Henry et al., 1982). Exemplary linkers for use in the invention have a combination of glycine, alanine, proline and methionine residues, such as AAAGGM (SEQ ID NO:19), AAAGGMPPAAAGGM (SEQ ID NO:20) and AAAGGM(PPAAAGGM₂ (SEQ ID NO:21). However, any flexible linker generally between about 6 and about 40 amino acids in length, or so, may be used. Linkers may have virtually any sequence that results in a generally flexible peptide, including alanine-proline rich sequences of the type exemplified above.

It also may be desirable to include additional sequences or "tags" not related to adenylyl cyclase function. For purposes of isolation, one may include in the construct a short peptide for which a binding partner is readily available. Examples of such fusion protein expression systems are the glutathione S-transferase system (Pharmacia, Piscataway, N.J.), the maltose binding protein system (NEB, Beverley, Mass.), the FLAG system (IBI, New Haven, Conn.) and the 6xHis system (Qiagen, Chatsworth, Calif.). Some of these systems produce fusions bearing only a small number of additional amino acids which are unlikely to affect the structure of the recombinant product.

For example, both the FLAG system and the 6xHis system add only short sequences, both of which are known to be poorly antigenic and which do not adversely affect folding of the protein to its native conformation. Indeed, the 6xHis fusion protein has already been generated and purified. This molecule has been purified to essential homogeneity by three sequential chromatographic steps--Qiagen Ni⁺ column; mono Q chromatography; phenyl superose chromatography. The purified fusion protein has a specific activity of 13 μg/min/mg, essentially the same as purified type I or type II adenylyl cyclase. The fusion protein is activated synergistically by G_(s)α and forskolin, and it is inhibited by G protein βγ subunits and P-site inhibitors, e.g., 2'-deoxy-3'-AMP.

With other fusion systems, it is desirable to excise the tag from the desired product. In a preferred embodiment, the tag is linked to the recombinant protein by a peptide sequence containing a specific recognition sequence for a protease. Examples of suitable sequences are those recognized by the Tobacco Etch Virus protease (Life Technologies, Gaithersburg, Md.) or Factor Xa (New England Biolabs, Beverley, Mass.).

More subtle changes may be made in the amino acid sequences provided herein while retaining a molecule having appropriate structure and function. For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of structural and functional integrity. These "conservative" changes are defined herein as "equivalent" in terms of this application.

Conservative amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. An analysis of the size, shape and type of the amino acid side-chain substituents reveals that arginine, lysine and histidine are all positively charged residues; that alanine, glycine and serine are all a similar size; and that phenylalanine, tryptophan and tyrosine all have a generally similar shape. Therefore, based upon these considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine; are defined herein as constituting conservatively related groups, respectively.

In making such changes, the hydropathic index of amino acids also may be considered. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte & Doolittle, 1982, incorporated herein by reference). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with biological properties of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).

In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

Numerous scientific publications have been devoted to the prediction of secondary structure, and to the identification of epitopes, from analyses of amino acid sequences (Chou & Fasman, 1974a,b; 1978a,b; 1979). Any of these may be used, if desired, to supplement the teachings of Hopp in U.S. Pat. No. 4,554,101. In the instant situation, it should be even easier to predict which substitutions will be tolerated given that the primary (and predicted secondary) structures for numerous other adenylyl cyclases are known.

Two designations for amino acids are used interchangeably throughout this application, as is common practice in the art. Alanine=Ala (A); Arginine=Arg (R); Aspartate=Asp (D); Asparagine=Asn (N); Cysteine=Cys (C); Glutamate=Glu (E); Glutamine=Gln (Q); Glycine=Gly (G); Histidine=His (H); Isoleucine=Ile (I); Leucine=Leu (L); Lysine=Lys (K); Methionine=Met (M); Phenylalanine=Phe (F); Proline=Pro (P); Serine=Ser (S); Threonine=Thr (T); Tryptophan=Trp (W); Tyrosine=Tyr (Y); Valine=Val (V).

                  TABLE 2                                                          ______________________________________                                         Amino Acid     Hydropathic Index                                               ______________________________________                                         Isoleucine     4.5                                                               Valine 4.2                                                                     Leucine 3.8                                                                    Phenylalanine 2.8                                                              Cysteine/cystine 2.5                                                           Methionine 1.9                                                                 Alanine 1.8                                                                    Glycine -0.4                                                                   Threonine -0.7                                                                 Tryptophan -0.9                                                                Serine -0.8                                                                    Tyrosine -1.3                                                                  Proline -1.6                                                                   Histidine -3.2                                                                 Glutamic Acid -3.5                                                             Glutamine -3.5                                                                 Aspartic Acid -3.5                                                             Asparagine -3.5                                                                Lysine -3.9                                                                    Arginine -4.5                                                                ______________________________________                                    

                  TABLE 3                                                          ______________________________________                                         Amino Acid    Hydrophilic Index                                                ______________________________________                                         arginine      +3.0                                                               lysine +3.0                                                                    aspartate +3.0 ± 1                                                          glutamate +3.0 ± 1                                                          serine +0.3                                                                    asparagine +0.2                                                                glutamine +0.2                                                                 glycine 0                                                                      threonine -0.4                                                                 alanine -0.5                                                                   histidine -0.5                                                                 proline -0.5 ± 1                                                            cysteine -1.0                                                                  methionine -1.3                                                                valine -1.5                                                                    leucine -1.8                                                                   isoleucine -1.8                                                                tyrosine -2.3                                                                  phenylalanine -2.5                                                             tryptophan -3.4                                                              ______________________________________                                    

Nucleic acid sequences of the present invention also may be varied from the exemplified sequences of SEQ ID NO:17 or SEQ ID NO:1, 3, 5, 7, 9, 11, 13 or SEQ ID NO:15. Due to the degeneracy of the genetic code, multiple codons can encode a single amino acid. Thus, different nucleic acids may encode the same polypeptide. By reference to the following chart, any codon may be substituted for a corresponding codon:

                  TABLE 4                                                          ______________________________________                                         Amino Acids    Codons                                                          ______________________________________                                         Alanine   Ala    A     GCA  GCC  GCG  GCU                                        Cysteine Cys C UGC UGU                                                         Aspartic acid Asp D GAC GAU                                                    Glutamic acid Glu E GAA GAG                                                    Phenylalanine Phe F UUC UUU                                                    Glycine Gly G GGA GGC GGG GGU                                                  Histidine His H CAC CAU                                                        Isoleucine Ile I AUA AUC AUU                                                   Lysine Lys K AAA AAG                                                           Leucine Leu L UUA UUG CUA CUC CUG CUU                                          Methionine Met M AUG                                                           Asparagine Asn N AAC AAU                                                       Proline Pro P CCA CCC CCG CCU                                                  Glutamine Gln Q CAA CAG                                                        Arginine Arg R AGA AGG CGA CGC CGG CGU                                         Serine Ser S AGC AGU UCA UCC UCG UCU                                           Threonine Thr T ACA ACC ACG ACU                                                Valine Val V GUA GUC GUG GUU                                                   Tryptophan Trp W UGG                                                           Tyrosine Tyr Y UAC UAU                                                       ______________________________________                                    

For convenience in cloning, it also may be desirable to alter the nucleic acid sequence to create or delete restriction enzyme sites. In some instances, this may alter the amino acid sequence of the resulting protein product. It is expected that these alterations will be tolerated without the loss of function given their presence at the ends of the catalytic domains. Though some changes conceivably could impair function, these can readily be determined by screening in recombinant hosts, as described below.

In other embodiments it is desired that the C₁ and C₂ domains of mammalian adenylyl cyclases are separately expressed and reconstituted into a simple mixture. The inventors have demonstrated that it is possible to simply admix separately synthesized cytosolic domains C₁ and C₂ of adenylyl cyclases and retrieve adenylyl cyclase activity when no such activity exists in the cytosolic domains separately. Hence, similarly to the fusion chimeras discussed above it would be a simple matter to admix any of the 64 possible combinations of C₁ and C₂ regions to determine which provide functional enzyme composition (Table 1).

As with the fusion proteins of the present invention the C₁ and C₂ complexes, are activated synergistically by G_(s)α and forskolin, and inhibited by G protein βγ subunits and P-site inhibitors, e.g., 2'-deoxy-3'-AMP.

These observations make it clear that there is cooperativity between the C_(1a) and the C_(2a) domains for catalytic activity to occur. Furthermore the inventors have demonstrated that these two domains spontaneously interact with one another to form an active complex. Such a complex may be facilitated by a positive covalent interaction as exemplified by the chimeric compositions of the present invention. Alternatively, a complex between the C_(1a) and C_(2a) may form, in solution, due to non-covalent interactions, for example ionic interactions, between the two domains to yield an active adenylyl cyclase activity. By non-covalent interaction is meant any interaction that allows C₁ and a C₂ domain of adenylyl cyclase to interact and produce an adenylyly cyclase activity without a chemical bond linking the components of one domain with the components of the other.

2. Expression Vectors

Expression vectors are genetic constructs that encode gene products and sequences necessary for the expression thereof. A typical expression vector is a bacterial plasmid or phage, such as any of the pUC plasmid series, Bluescript™ or other commercially available, multipurpose cloning vehicles. Expression vectors for use in eukaryotic systems also exist and include integrative and non-integrative plasmids as well as viral vectors such as retrovirus, adenovirus, herpesvirus and baculovirus.

One of the universal structural features of expression vectors is the presence of regulatory elements that permit an inserted gene to be expressed when the vector is brought into contact with RNA- and protein-synthetic machinery. Expression vectors also typically have an origin of replication so that the vector can be propagated in the appropriate host system. In addition, most encode (i) a multipurpose cloning site for easy insertion of gene sequences and (ii) a selectable marker so that host cells carrying the vector can be selected from those that do not.

The expression vector also may include particular ribosome binding sites, polyadenylation sites or any other elements necessary for the expression of the DNA in a host cell. These elements, along with the aforementioned regulatory elements, are combined into expression vector constructs by methods well known and routinely practiced in the art such as restriction enzyme digestion followed by DNA ligase directed splicing of the various genetic elements.

The term "regulatory elements" is meant to include promoters. In prokaryotic systems, the promoter region is about 40 base pairs in length, starting about five to eight base pairs upstream of RNA initiation site (designated +1). Within this sequence is a region referred to as the Pribnow box, a six base pair motif centered about the -10 region. The consensus sequence is TATAAT. Another consensus sequence is about -35. This region is important for accurate initiation of transcription.

Promoters that are most commonly used in recombinant DNA construction include the β-lactamase (penicillinase), lactose and tryptophan (trp) promoter systems. While these are the most commonly used, other microbial promoters have been discovered and utilized, and details concerning their nucleotide sequences have been published, enabling those of skill in the art to ligate them functionally with plasmid vectors.

Eukaryotic promoters are composed of multiple genetic elements including that group of transcriptional control modules clustered around the initiation site for RNA polymerase II. These discrete functional modules each comprise approximately 7-20 bp of DNA and contain one or more recognition sites for transcriptional activator proteins. At least one module in each promoter functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.

Additional eukaryotic promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between some elements is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.

Preferred eukaryotic promoters are viral promoters such as the adenovirus major later promoter, SV40 late promoter from simian virus 40, the Baculovirus polyhedron enhancer/promoter element, Herpes Simplex Virus thymidine kinase (HSV tk), the immediate early promoter from cytomegalovirus (CMV) and various retroviral promoters including LTR elements. The elements are operably linked to a gene, the expression of which is desired. By "operably linked," it is meant that the regulatory element is positioned, relative to a coding sequence, such that expression of that coding sequences is effected by that element.

The promoter further may be characterized as an inducible promoter. An inducible promoter is a promoter which is inactive or exhibits low activity except in the presence of an inducer substance. Some examples of inducible promoters that may possibly be included as a part of the present invention include, but are not limited to, MT II, MMTV (mouse mammary tumor virus), Collagenase, Stromelysin, SV40, Murine MX Gene, α-2-Macroglobulin, MHC Class I Gene H-2kb, HSP70, Proliferin, Tumor Necrosis Factor or Thyroid Stimulating Hormone α Gene. It is understood that any inducible promoter may be used in the practice of the invention and that all such promoters would fall within the spirit and scope of the claimed invention.

The eukaryotic regulatory elements of the present invention also may comprise an enhancer, operably linked to the gene of interest. The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Aside from this operational distinction, enhancers and promoters are very similar. They have the same general function of activating transcription in the cell and often have overlapping, contiguous and seemingly similar modular organization.

Below is a list of enhancers that can be used in combination with the present constructs:

                                      TABLE 5                                      __________________________________________________________________________     ENHANCER       REFERENCES                                                      __________________________________________________________________________     Immunoglobulin Heavy Chain                                                                    Hanerji et al., 1983; Gilles et al., 1983; Grosschedl and                        Baltimore, 1985; Atchinson and Perry, 1986, 1987; Imler                        et al., 1987; Weinberger et al., 1988; Kiledjian et al.,         1988; Porton et al., 1990                                                     Immunoglobulin Light Chain Queen and Baltimore, 1983; Picard and                            Schaffner, 1984                                                   T-Cell Receptor Luria et al., 1987, Winoto and Baltimore, 1989; Redondo         et al., 1990                                                                  HLA DQ α and DQ β Sullivan and Peterlin, 1987                       β-Interferon Goodbourn et al., 1986; Fujita et al., 1987; Goodbourn        and Maniatis, 1985                                                            Interleukin-2 Greene et al., 1989                                              Interleukin-2 Receptor Greene et al., 1989; Lin et al., 1990                   MHC Class II 5 Koch et al., 1989                                               MHC Class II HLA-DRα Sherman et al., 1989                                β-Actin Kawamoto et al., 1988; Ng et al., 1989                            Muscle Creatine Kinase Jaynes et al., 1988; Horlick and Benfield, 1989;                     Johnson                                                            et al., 1989a                                                                 Prealbumin (Transthyretin) Costa et al., 1988                                  Elastase I Omitz et al., 1987                                                  Metallothionein Karin et al., 1987; Culotta and Hamer, 1989                    Collagenase Pinkert et al., 1987; Arigel et al., 1987                          Albumin Gene Pinkert et al., 1987, Tronche et al., 1989, 1990                  α-Fetoprotein Godbout et al., 1988; Campere and Tilghman, 1989                         τ-Globin Bodine and Ley, 1987; Perez-Stable and                           Constantini,                                                       1990                                                                          β-Globin Trudel and Constantini, 1987                                     e-fos Cohen et al., 1987                                                       c-HA-ras Triesman, 1986; Deschamps et al., 1985                                Insulin Edlund et al., 1985                                                    Neural Cell Adhesion Molecule Hirsch et al., 1990                              (NCAM)                                                                         α1-Antitrypsin Latimer et al., 1990                                      H2B (TH2B) Histone Hwang et al. 1990                                           Mouse or Type I Collagen Ripe et al., 1989                                     Glucose-Regulated Proteins Chang et al., 1989                                  (GRP94 and GRP78)                                                              Rat Growth Hormone Larsen et al., 1986                                         Human Serum Amyloid A (SAA) Edbrooke et al., 1989                              Troponin I (TN I) Yutzey et al., 1989                                          Platelet-Derived Growth Factor Pech et al., 1989                               Duchenne Muscular Dystrophy Klamut et al., 1990                                SV40 Banerji et al., 1981; Moreau et al., 1981; Sleigh and                      Lockett, 1985; Firak and Subramanian, 1986; Herr and                           Clarke, 1986; Imbra and Karin, 1986; Kadesch and Berg,                         1986; Wang and Calame, 1986; Ondek et al., 1987; Kuhl                          et al., 1987 Schaffner et al., 1988                                           Polyoma Swartzendruber and Lehman, 1975; Vasseur et al., 1980;                  Katinka et al., 1980, 1981; Tyndell et al., 1981; Dandolo                      et al., 1983; deVilliers et al., 1984; Hen et al., 1986;                       Satake et al., 1988; Campbell and Villarreal, 1988                            Retroviruses Kriegler and Botchan, 1982, 1983; Levinson et al., 1982;                         Kriegler et al., 1983, 1984a,b, 1988; Bosze et al.,                          1986;                                                              Miksicek et al., 1986; Celander and Haseltine, 1987;                           Thiesen et al., 1988; Celander et al., 1988; Chol et al.,                      1988; Reisman and Rotter, 1989                                                Papilloma Virus Campo et al., 1983; Lusky et al., l983; Spandidos and                         Wilkie, 1983; Spalholz et al., 1985; Lusky and Botchan,                        1986; Cripe et al., 1987; Gloss et al., 1987; Hirochika                      et                                                                 al., 1987, Stephens and Hentschel, 1987; Glue et al.,                          1988                                                                          Hepatitis B Virus Bulla and Siddiqui, 1986; Jameel and Siddiqui, 1986;                        Shaul and Ben-Levy, 1987; Spandau and Lee, 1988;                               Vannice and Levinson, 1988                                      Human Immunodeficiency Virus Muesing et al., 1987; Hauber and Cullan,                       1988;                                                              Jakobovits et al., 1988; Feng and Holland, 1988; Takebe                        et al., 1988; Rowen et al., 1988; Berkhout et al., 1989;                       Laspia et al., 1989; Sharp and Marciniak, 1989;                                Braddock et al., 1989                                                         Cytomegalovirus Weber et al., 1984; Boshart et al, 1985; Foecking and                         Hofstetter, 1986                                                Gibbon Ape Leukema Virus Holbrook et al., 1987; Quinn et al., 1989           __________________________________________________________________________

3. Host Cells and Expression Systems

Once an expression vector has been generated, it is necessary to provide the transcriptional and translational machinery that will facilitate expression of the gene encoded by the vector. Such expression systems are well known to the skilled practitioner in the art and include bacterial systems such as E. coli, yeast systems such as Pichia pastoris, the insect system derived from baculovirus and various mammalian expression systems such as COS or CHO cells.

In a preferred embodiment, polypeptides are expressed in E. coli systems. A selected adenylyl cyclase gene construct is inserted into an prokaryotic expression vector by standard subcloning techniques and an E. coli expression host transformed (Dessauer and Gilman, 1996). Recombinant E. coli are grown in any of a number of suitable media, for example LB, to effect the expression of the adenylyl cyclase. After culturing the bacteria for a sufficient period of time, to be optimized by the operator, the cells are collected by centrifugation and washed to remove residual media. In certain embodiments, the polypeptides expressed in this system are fusion proteins containing the C₁ and the C₂ regions. In other aspects of the present invention, the C₁ and the C₂ domains are co-expressed within the same host system. In yet another embodiment, the polypeptides are separately expressed in distinct expression hosts and the C₁ and C₂ polypeptides are isolated and combined after that expression.

Cells are lysed, for example, by disruption in a cell homogenizer, and centrifuged to separate and cell membranes from the soluble cell components. This centrifugation can be performed under conditions whereby inclusion bodies, if present, are selectively enriched by incorporation of sugars such as sucrose into the buffer and centrifugation at a predetermined speed.

Soluble adenylyl cyclase or the components of the adenylyl cyclase complex (the C_(1a) and C_(2a) domains) may be purified from host cells according to standard methodology. See, for example, Harris et al. (1989). For example, cell extracts may be purified by ammonium sulfate precipitation, ion exchange chromatography, sequential size exclusion chromatography, isoelectric focusing, HPLC size exclusion chromatography, ultracentrifugation or ultrafiltration. Alternatively, incorporated sequences tags may serve as immunological binding partners in antibody-based affinity purification protocols, as described herein.

If the recombinant adenylyl cyclase is expressed as inclusion bodies, as is the case in many instances, these can be washed in any of several solutions to remove some of the contaminating host proteins, then solubilized in solutions containing high concentrations of urea (e.g. 8M) or chaotropic agents such as guanidine hydrochloride in the presence of reducing agents such as β-mercaptoethanol or DTT (dithiothreitol).

Under some circumstances, it may be advantageous to incubate the protein for several hours under conditions suitable for the protein to undergo a refolding process into a conformation which more closely resembles that of the native protein. Such conditions generally include low protein concentrations less than 500 μg/ml, low levels of reducing agent, concentrations of urea less than 2 M and often the presence of reagents such as a mixture of reduced and oxidized glutathione which facilitate the interchange of disulfide bonds within the protein molecule.

The refolding process can be monitored, for example, by assay of enzymatic activity. Following refolding, the protein can then be purified further and separated from the refolding mixture by chromatography on any of several supports including ion exchange resins, gel permeation resins or on a variety of affinity columns. Storage at ultra-low temperatures (e.g., -80° C.) is recommended.

In another embodiment, the expression system is derived from the insect virus baculovirus. The gene encoding the polypeptide can be manipulated by standard techniques in order to facilitate cloning into the baculovirus vector such that it is under the control of the powerful polyhedron promoter. See Ausubel et al., supra. A preferred baculovirus vector is the pBlueBac vector (Invitrogen, Sorrento, Calif.). The vector carrying the gene for the polypeptide is transfected into Spodoptera frugiperda (Sf9) cells by standard protocols and the cells are cultured and processed to produce the recombinant antigen. See Summers et al. (1987); U.S. Pat. No. 4,215,051, incorporated herein by reference.

Although baculovirus expression in insect cells is generally a well known technique, one may additionally refer to any one of the various scientific articles that have been published concerning the use of the baculovirus system specifically in the expression of adenylyl cyclase. By way of example only, Tang et al. (1991), Gao & Gilman (1991) and Taussig et al. (1995) are each incorporated herein by reference for the purposes of describing in even more detail the expression of adenylyl cyclase in Sf9 cells.

In embodiments where it is desirable to combine the C₁ and C₂ peptides in solution it is understood that the domains are admixed in a suitable buffer that allows for adenylyl cyclase activity to be determined. Such assay and buffer conditions are described in the literature and are well within the skill of the ordinary person skilled in the art (Smigel 1986).

4. Screening of Compounds for Effects on Adenylyl Cyclase Activity

It will be useful, for a variety of clinical indications, to identify inhibitors and stimulators of adenylyl cyclase activity. For example, inhibitors are useful in the treatment of cholera, pituitary tumors, heart failure, ischemia and certain endocrine diseases. As persistent stimulation of the β-adrenergic pathway in cardiocytes can lead to cell necrosis, an inhibitor of this pathway downstream of the receptor would be useful in treating patients with heart conditions. Stimulation of adenylyl cyclase could be useful in the treatment of pseudohypoparathyroidism or other endocrine deficiencies.

The soluble adenylyl cyclase compositions of the present invention may advantageously be used to assess the inhibitory or stimulatory effects of drugs on adenylyl cyclase activity.

Purified or crude enzyme, as described above, can be employed in assays in vitro to assess the ability of particular agents to affect adenylyl cyclase activity. Salomon et al. (1974); Tang et al. (1991); Taussig et al. (1994) are also incorporated herein by reference for the purpose of describing adenylyl cyclase assays in even more detail.

In an alternative embodiment it is possible to create positive selection systems that permit the identification of inhibitors and stimulators of adenylyl cyclase activity without the need for purifying soluble adenylyl cyclase. Specifically, by making host cell growth dependent upon the stimulation of adenylyl cyclase, it is possible to simply treat host cells expressing adenylyl cyclase with a putative inhibitor or stimulator and assess the effects by measuring growth of the host. Another kind of positive selection relies on phenotypic changes in the host when adenylyl cyclase is expressed, such as a colorimetric indication.

A host cell was engineered such that it is dependent upon the synthesis of a heterogeneous adenylyl cyclase. The E. coli strain Δcya TP2000 lacks adenylyl cyclase activity and, therefore, cannot utilize maltose as a carbon source and cannot grow on minimal essential media (minimal essential media). When transformed with the soluble adenylyl construct of the present invention, along with a G-protein construct for activation of the adenylyl cyclase, the recombinant host regained the ability to utilize maltose and grown on MEM.

Inhibitors of adenylyl cyclase are readily identified by incubation with the recombinant host described above. When a substance inhibits adenylyl cyclase, the growth of the host will be reduced when compared to that of a control culture not treated with the substance. Similarly, the ability to utilize maltose is measured by growth on McConkey agar; a positive result is indicated by red coloration of adenylyl cyclase⁺ colonies. Where substance inhibits adenylyl cyclase activity, the coloration will be reduced or eliminated.

Stimulators of adenylyl cyclase also can be readily identified using the same recombinant strain identified above. By looking for increases in growth on MEM or increases in intensity of coloration on McConkey agar, when compared to untreated control cultures, increased adenylyl cyclase activity is identified. The rapidity of growth and coloration also is an indicator of stimulation.

It will, of course, be recognized that the particular benefits of the screening assays of the present invention may lie in the identification of candidate substances as a starting point for developing a therapeutic product, and that further modification of the substances identified may be desired. For example, in order to achieve optimal, or improved, stimulation or inhibition, or to reduce any toxicity, or such like.

5. Example

Even though the invention has been described with a certain degree of particularity, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the foregoing disclosure. Accordingly, it is intended that all such alternatives, modifications, and variations which fall within the spirit and the scope of the invention be embraced by the defined claims.

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1 Construction of Type I Cytoplasmic Domain C₁α

To produce DNA for expression of IC₁, restriction enzyme sites for NcoI and NotI were introduced at nucleotide 704 (amino acid residue 237 of type I adenylyl cyclase) and nucleotide 1453 (amino acid residue 484 of type I adenylyl cyclase), respectively, and an internal NcoI site was eliminated by two rounds of mutagenesis using M13-mp18-C₁ as the template (Kunkel et al., 1987). The 0.7 kB NcoI-EcoRI fragment was cloned into the same sites of pTrcl-lisA (prokaryotic expression vector from Invitrogen, San Diego, Calif.), resulting in pTrc-IsC₁. A termination site was introduced by adding phosphorylated linkers (5'-GGCCGCTCACCATCACCATCACCATTAGG (SEQ ID NO:22) and 5'-AATTCCTAATGGTGATGGTGATGGTGAGA (SEQ ID NO:23)) to pTro-IsC₁ that had been digested with NotI and EcoRI; the resulting plasmid was used for expression of IC₁.

EXAMPLE 2 Construction of Type II Cytoplasmic Domain C₂α

To produce DNA for expression of IIC₂, a 0.9 kB SspI-KpnI fragment was isolated from pSK-rACII (pBluescript with a cDNA insert that encodes type II adenylyl cyclase). This fragment was ligated with phosphorylated linkers (5'-GATCCATCATGAGACAGAGTGAAT (SEQ ID NO:24) and 5'-ATTCACTCTGTCTCATGATC(SEQ ID NO:25)) and pUC18 that had been digested with BamHI and KpnI, resulting in pUC-IIC₂. The 0.9 kB BspHI-EcoRI fragment from pUC-IIC₂ was transferred to pTrc-HisA that had been digested with NcoI and EcoRI for expression of IIC₂ (residues 821 to 1090 of type II adenylyl cyclase).

EXAMPLE 3 Construction of Covalent Chimeras of C₁α and C₂α

To link IC₁ and IIC₂, the 0.9 kB BspHI-EcoRI fragment from pUC-IIC₂ was ligated with phosphorylated linkers (5'-GGCCGCTGGAGG (SEQ ID NO:26) and 5'-GATGCCTCCAGC (SEQ ID NO:27)) and pTrc-IaC₁, that had been digested with NotI and EcoRI. One, three or five sets of linkers were incorporated, resulting in pTrc-IC₁ IIC₂ -L₁, pTrc-IC₁ IIC₂ -L₃, and pTrc-IC₁ IIC₂ -L₅, respectively. A small deletion (56 base pairs) at the sequence encoding the amino terminus of IC₁ IIC₂ (right after the NcoI site) occurred during subcloning. The site of initiation of IC₁ IIC₂ -L₃ is thus residue 271. To express G_(s)α, a 1.3-kB NcoI (blunted) to HinDIII fragment encoding either G_(s)α-1 or the Gln²²⁷ -Leu mutant of G_(s)α-1 was ligated with the 4.5 kB NcoI (blunted) to EcoRI fragment from pBB131 (Knoll and Gordon, 1993).

Concurrent expression of the NH₂ -terminal half of type I adenylyl cyclase and the carboxy-terminal half of type II adenylyl cyclase results in the formation of a noncovalent chimera that remains sensitive to both G.sub.α and forskolin, despite extremely low basal activity. The inventors have thus ligated cDNA's that encode C_(1a) from type I adenylyl cyclase and C_(2a) from type II adenylyl cyclase with short linkers between them, resulting in constructs designated IC₁ IIC₂ -L₁, IC₁ IIC₂ -L₃ and IC₁ IIC₂ -L₅ (FIG. 1).

EXAMPLE 4 Characteristics and Activity of Covalent Chimeras

E. coli that contained the desired plasmids were grown in LB plus 50 μM carbenicillin to OD₆₀₀ =0.3. Isopropyl-β-D-thiogalactopyranoside (100 μM) and chloramphenicol (0.5 μM) were added to induce expression of adenylyl cyclase for 12 hours. Bacteria were then collected by centrifugation at 4° C. and lysed by incubation at 4° C. for 30 min in 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 2 mM dithiothreitol (DTT) and protease inhibitors with 0.1 mg/ml of lysozyme. The suspension was sonicated briefly (3×20 sec) during incubation. The lysate was centrifuged (4° C.) at 150,000×g for 30 min, and the supernatant was recovered.

To evaluate the capacity of these constructs to encode functional adenylyl cyclases, the inventors tested complementation of the catabolic defect in Escherichia coli Δcya TP2000 which lacks adenylyl cyclase activity (Roy and Danchin, 1982). This deficient bacterial strain cannot utilize maltose as a carbon source; colonies thus fail to turn red on McConkey agar and cannot grow on minimal medium (Perlman and Pastan, 1969). To activate adenylyl cyclase in E. coli, the inventors coexpressed the cyclase constructs with either the wild-type alpha subunit of the G protein (G_(s)α) or a mutant G_(s)α (in which Gln²²⁷ is replaced with Leu; designated G.sub.α *) that is deficient in guanosine triphosphatase (GTPase) activity and, thus, is constitutively active (Graziano and Gilman, 1989).

E. coli TP2000 were transformed with two compatible plasmids--one for the expression of G_(s)α and the other for expression of the adenylyl cyclase constructs. Transformants were selected for resistance to carbenicillin and kanamycin and bacteria were cultured on either McConkey or M63 agar containing 0.4% maltose, 50 μM carbenicillin, 50 μM kanamycin and 100 μM isopropyl-β-D-thiogalactopyranoside. Cells were grown at 30° C. for the indicated times. Forskolin (5 μl, 10 mM) was spotted on the plates where indicated (+Fsk) before addition of transformants.

E. coli TP2000 expressing either G_(s)α or G_(s)α * remained pale yellow on McConkey agar supplemented with maltose and failed to grow on minimal medium (M63 medium containing arginine and maltose). However, bacteria expressing G_(s)α * and any of the three chimeric adenylyl cyclase constructs turned red on McConkey agar and grew on minimal medium. Wild-type E. coli turn red in about one half the time required for the chimeric constructs. Correction of the catabolic defect also was evident when wild-type G_(s)α was expressed with IC₁ IIC₂ -L₃ or IC₁ IIC₂ -L₅, but longer times were required. There was evidence of a small amount of cyclic AMP synthesis when construct IIC₂ was coexpressed with G_(s)α *, but no such effect was seen with IC₁. Similar ligation of C_(1a) and C_(2a) from type I adenylyl cyclase failed to produce a functional enzyme by these criteria, however.

The inventors also tested four mutants of G_(s)α that are altered in positions corresponding to the α3-β5 and α4-β6 loops of G.sub.α and G.sub.α1 and the α3 helix (Noel et al., 1993; Coleman et al., 1994). These mutants have a reduced ability to activate adenylyl cyclase (Berlot and Bourne, 1992). The cDNA's encoding these proteins also were altered to substitute Cys for Arg²⁰¹. This mutation also inhibits GTPase activity and activated the a subunit. These cDNA's were transferred into an expression vector and tested their ability to activate IC₁ IIC₂ -L₃ in E. coli. As a control, E. coli Δcya turned red on McConkey agar when transformed with vectors encoding G.sub.α Arg²⁰¹ →Cys and IC₁ IIC₂ -L₃. Under the same conditions, G_(s)α with a mutation in the α3 helix failed to show activity, whereas the other three mutants were indistinguishable from the control protein. These results correlate well with those obtained by transient expression of the same proteins in HEK 293 cells.

The inventors examined adenylyl cyclase activity in vitro in 150,000 g supernatant fractions from E. coli T2000 transformed with the various constructs. The soluble fraction from cells containing a control plasmid had no detectable adenylyl cyclase activity (FIG. 2). By contrast, supernatants from cells expressing IC₁ IIC₂ -L₁, IC₁ IIC₂ -L₃, IC₁ IIC₂ -L₅ displayed basal adenylyl cyclase activity (about 2 pmol/min/mg protein) that was activated by 200 nM G_(s)α bound to GTP-γ-S(50-fold), 100 μM forskolin (150 to 200-fold) or a combination of the two (600-fold). Ca²⁺ -calmodulin had no detectable effect on activity, and lysates from cells expressing either IC₁ or IIC₂ had little adenylyl cyclase activity.

The expected 60 kD protein was detected in appropriate E. coli supernatants with an antiserum to the carboxy-terminus of type II adenylyl cyclase (antiserum C2-1077), although the signal was not strong. Supernatants (60 μg) were alkylated with N-ethylmaleimide, resolved by SDS-polyacrylamide gel electrophoresis (PAGE) (11% gels), transferred to nitrocellulose and stained with affinity purified antiserum C2-1077 directed against the carboxy-terminus of type II adenylyl cyclase. The appropriate 29 kD soluble protein was present in cells expressing IIC₂. Also detected were 32 kD, 34 kD and 36 kD proteins in cells expressing IC₁ IIC₂ -L₁, IC₁ IIC₂ -L₃ and IC₁ IIC₂ -L₅, respectively. Other smaller proteins were present in extracts from all cells containing DNA for IIC₂. These proteins may arise from proteolysis or initiation of translation from downstream sites.

The adenylyl cyclase activity in 150,000 g supernatants from cells expressing IC₁ IIC₂ -L₃ was activated by addition of either forskolin (FIG. 3) or GTP-γ-S-G.sub.α (FIG. 4). The median effective concentration (EC₅₀) for forskolin was about 7 μM. An analog, 1,9-dideoxyforskolin, which does not activate mammalian adenylyl cyclases (Seamon et al., 1983), also failed to stimulate this enzyme. The EC₅₀ for activation of IC₁ IIC₂ -L₃ by GTP-γ-S-G.sub.α was about 1 μM, a value 20 to 50 times greater than that observed with type I or type II adenylyl cyclase. However, the maximal stimulatory effect of the G protein α subunit exceeded that of forskolin (FIG. 4). The stimulatory effects of minimally effective concentrations of GTP-γ-S-G.sub.α and forskolin were synergistic (FIG. 5), which also is characteristic of several mammalian adenylyl cyclases (Sutkowski et al., 1994). When IC₁ IIC₂ -L₃ was activated with forskolin or GTP-γ-S-G.sub.α, values of the Michaelis constants for ATP (K_(m),ATP) were 0.44 and 0.11 mM, respectively (FIG. 6). Addition of GTP-γ-S-G.sub.α in the presence of forskolin did not change the K_(m). Synergistic activation of the enzyme was thus not due to alternation of apparent substrate affinity.

Forskolin regulates the functions of a number of intrinsic membrane proteins including adenylyl cyclases (Seamon and Daly, 1986), glucose transporters (Kashiwagi et al., 1983; Joost et al., 1988), voltage-gated potassium channels (Hoshi et al., 1988), nicotinic cholinergic receptors (Wagoneer and Pallotta, 1988), a GABA receptor (Heuschneider and Schwartz, 1989) and P glycoproteins (Wadler and Wiernik, 1988; Morris et al., 1991). These proteins share no obvious amino acid sequence homology. However, all do have one or more hydrophobic domains predicted to include four or six transmembrane helices, and forskolin is highly lipophilic. Attempts to map forskolin binding sites have implicated the transmembrane helices or residues immediately adjacent to these domains (Wadzinski et al., 1990; Morris et al., 1994). It thus was surprising to detect activation of the IC₁ IIC₂ constructs by forskolin. Perhaps the interaction of forskolin with adenylyl cyclase is different from that with other proteins. Although forskolin activates adenylyl cyclases, it inhibits or blocks the pore conductivity of the other forskolin-regulated proteins.

The G protein βγ subunit complex (to 1 μM) inhibited the chimeric adenylyl cyclase while myristylated GTP-γ-S-G.sub.α1 (2 μM) had little effect on the basal or stimulated activity of IC₁ IIC₂ -L₃. Forskolin-activated IC₁ IIC₂ -L₃ was inhibited noncompetitively by 2'-deoxy-3'-AMP (a so-called "P-site" inhibitor) (FIG. 7). The enzyme was most sensitive to inhibition by the P-site analog when it was maximally stimulated by both forskolin and GTP-γ-S-G.sub.α. These properties are characteristic of P-site inhibition of mammalian adenylyl cyclases (Johnson and Shoshani, 1990; Florio and Ross, 1983).

A supernatant containing IC₁ IIC₂ -L₃ was subjected to gel filtration through Pharmacia Superdex 75. A major peak of adenylyl cyclase activity consistent with a globular 60 kD protein was observed, along with a minor peak of about twice the size (FIG. 8). The soluble fraction (200 μl) from E. coli expressing IC₁ IIC₂ -L₃ was applied to a Pharmacia Superdex 75 HR 10/30 gel filtration column that had been equilibrated with 20 mM Tri-HCl (pH 8.0), 1 MM EDTA, 2 mM DTT and 500 mM NaCl. The flow rate was 0.3 ml/min and 0.3 ml fractions were collected. Adenylyl cyclase activity was measured in the presence of 10 mM MgCl₂ and 100 μM forskolin. Portions of selected fractions were subjected to SDS-PAGE and immunoblotting. The active enzyme thus appears to migrate as a monomer, although some may be present as dimers. The 60 kD immunoreactive band was present within the major peak of adenylyl cyclase activity, whereas the 27 kD and 34 kD bands were not. Proteolysis was evident in these extracts. Further chromatography of the material shown in FIG. 8 on a Pharmacia mono Q column revealed multiple peaks of activity, and only a fraction of the active enzyme was recognized by antiserum C2-1077 (directed against the COOH-terminus).

The 60 kD protein has been purified to near homogeneity. These preparations are devoid of other immunoreactive bands. It is thus clear that the 60 kD protein is the active species. The turnover number of the purified protein is close to the value for purified type II adenylyl cyclase.

EXAMPLE 5 Adenylyl Cyclase Activity in Mixtures of Cytosolic Domains

Materials and Methods

DNA Constructs, Antibodies, and G Protein Subunits. To create the DNA for expression of the C_(1a) domain of type I adenylyl cyclase (IC₁), the construct pTrc(271)Ic₁ IIC₂ L3 (Tang and Gilman, 1995) was digested with BsrBI and ligated with the phosphorylated oligonucleotides 5'-GATCTAGCTAGCTA (SEQ ID NO:28) and 5'-TAGCTAGCTA (SEQ ID NO:29). The DNA was then digested with BspHI and BglII and ligated into pTreH6 (Dessauer and Gilman, 1996) that had been digested with NcoI and BglII. This resulted in a construct with an amino terminal hexa-histidine tag linked to residues 271-484 of type I adenylyl cyclase.

To create the DNA for expression of the C₂ domain of type II adenylyl cyclase (IIC₂), a polymerase chain reaction was performed on pTrc(271)IC₁ IIC₂ L3 using the primers 5'-ATGAGATCTGGATGCCAAGTTGCTCTGAG (SEQ ID NO:30) and 5'-TGGAGTCATGACACAGAGTGAAT (SEQ ID NO:31); this created an amino terminal BspHI restriction site and a carboxy terminal BglII restriction site. After excision with BspHI and BglII, this fragment was ligated into pQE60 (Qiagen, Chatsworth, Calif.) that had been digested with NcoI and BglII. This created a construct encoding residues 821-1090 of type II adenylyl cyclase with a hexa-histidine tag at the carboxy terminus.

The antibodies utilized in this work have been described (Dessauer and Gilman, 1996). G_(s)α was purified and activated with guanosine 5'-[γ-thio] triphosphate (GTP[γS]) as described (Dessauer and Gilman, 1996).

Expression of Proteins in E. coli. The IC₁ construct was transformed into E. coli strain BL21(DE3), and the cells were grown in the presence of ampicillin (50 μg/ml). The IIC₂ construct was transformed into BL21(DE3) cells also harboring the pREP4 plasmid; cells were grown in ampicillin (50 μg/ml). Cultures were grown to OD₆₀₀ =0.4 at 30° C.; isopropyl β-D-thiogalactoside (30 μM) was then added and cells were grown at room temperature for 15 h before harvesting and freezing in liquid nitrogen. Cell pellets were resuspended with a Polytron homogenizer in 1/15th the culture volume of lysis buffer (50 mM Tris.HCl, pH 8.0/10 mM 2-mercaptoethanol/50 mM NaCl) containing mixed protcase inhibitors (Dessauer and Gilman, 1996). Cells were lysed by addition of 0.2 mg/ml of lysozyme. After incubation for 30 min At 4° C., DNase was added (0.02 mg/ml plus 5 mM MgCl₂). This suspension was centrifuged at 100,000×g for 30 min, and the clarified lysate was collected.

Protein Purification. Clarified lysate from a 10-liter culture was supplemented with NaCl (250 mM final concentration) and loaded onto a 5-ml nickel-nitrilotriacetic acid (Ni²⁺ -NTA) column (Qiagen) that had been equilibrated with lysis buffer. The column was washed with 15 volumes of lysis buffer supplemented with 2 mM MgCl₂, 400 mM NaCl (final concentration), and 5 mM imidazole; 12 volumes of 50 mM Tris.HCl (pH 8.0), 10 mM 2-mercaptoethanol, 2 mM MgCl₂, and 15 mM imidazole; and 8 volumes of 50 mM Tris.HCl (pH 8.0), 10 mM 2-mercaptoethanol, 10 mM NaCl, and 15 mM imidazole. The column was then eluted with 8 volumes of 50 mM Tris-HCl (pH 8.0), 10 mM 2-mercaptoethanol, 10 mM NaCl, 2 mM MgCl₂, and 150 mM imidazole.

The Ni²⁺ -NTA column eluate containing IC₁ was adjusted to 400 mM ammonium sulfate and loaded directly onto a 2-ml phenyl Sepharose column that had been equilibrated in buffer A (50 mM Na-Hepes, pH 8.0/2 mM MgCl₂ /1 mM EDTA/2 mM dithiothreitol) plus 400 mM (NH₄)₂ SO₄. The Column was washed with 10 ml of equilibration buffer and 10 ml of buffer A, followed by elution with a linear gradient (20 ml) of 0 to 10 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) in buffer A and 20 ml of buffer A containing 10 mM CHAPS. The pooled peak was exchanged into buffer A and concentrated to 0.4 ml (≈1 mg/ml).

The Ni²⁺ -NTA column eluate containing IIC₂ was loaded directly onto a 8-ml Mono-Q 10/10 column (Pharmacia), equilibrated in buffer A, and washed with 5 volumes of the same buffer. Protein was eluted with a 120-ml linear gradient of NaCl (0-300 mM) in buffer A, followed by a steep gradient to 1 M NaCl. The IIC₂ protein represents >90% of the total loaded onto the column and elutes at ≈150 mM NaCl. This pooled material was exchanged into buffer A containing 50 mM NaCl and concentrated to 10 mg/ml.

Gel filtration was carried out on a Superdex 200 column (Pharmacia) in 50 mM Na-Hepes (pH 8.0), 2 mM MgCl₂, 1 mM EDTA, 2 mM dithiothreitol, and 150 mM NaCl. All samples were loaded onto the column in less than 0.5 ml.

Adenylyl Cyclase Assays. Adenylyl cyclase activity was quantified as described by Smigel (Smigel, 1986). All assays contained 10 mM MgCl₂ in a 50-μl final volume. Incubations were for 10 min at 30° C. unless otherwise indicated.

Results

The mixture of two crude bacterial lysates, each containing either the C₁ or the C₂ domains of type I and type II adenylyl cyclase, respectively, permits observation of GTP[γS]]-G_(s)α - and FSK-stimulated adenylyl cyclase activity (FIG. 9). Similar results were obtained when the C1 domain of type V adenylyl cyclase was mixed with the C2 domain of type II adenylyl cyclase. There was no detectable activity when IC₁, IIC₂ or VC1 was assayed alone in the presence or absence of activators. (The same result was obtained after purification of these fragments.) Based on this result, an assay was defined to facilitate purification of each fragment (FIG. 10). Lysates containing the proteins were purified by Ni²⁺ -NTA column chromatography as described under materials and methods. A fixed amount of this partially purified material was used as a reagent to assay increasing amounts of the complementary fragment in the presence of 50 μM FSK. Addition of IC₁ to a fixed amount of IIC₂ resulted in a roughly linear increase in enzymatic activity (FIG. 10A). Given the smaller amounts of IC₁ in these preparations, saturation was observed when increasing amounts of IIC₂ were added to the partially purified preparation of IC₁ (FIG. 10B). Assays were carried out in the linear ranges of these titrations.

Large amounts (6 mg/liter of culture) of the IIC₂ fragment were readily purified to homogeneity by Ni²⁺ -NTA and Mono Q column chromatography (FIG. 11 and Table 6). The IC₁ and IIC₂ were resolved on 11% polyacrylamide gels and stained with Coomassie blue, the material appeared to be homogeneous after SDS/PAGE. Amino acid sequencing revealed that the amino terminus of the protein began at Met-847 of type II adenylyl cyclase, 27 residues downstream from the presumed initiator methionine in the construct. Electron spray mass spectroscopy revealed a single species with a M_(r) of 28,258, consistent with a protein containing Met-847-Ser-1090 (calculated M_(r) =28,256). Gel filtration of this material showed a single peak of activity with an apparent M_(r) of ≈50,000 (FIG. 11). This behavior may represent dimerization of the fragment. The protein yield from these studies was 0.22 mg for IC₁, 57 mg for IIC₂ (Table 6). Similar studies with VC₁ yielded 10 mg of VC1 protein.

                  TABLE 6                                                          ______________________________________                                         PURIFICATION OF THE IC.sub.1 AND IIC.sub.2 FRAGMENTS                             OF MAMMALIAN ADENYLYL CYCLASE                                                                                       Specific                                     Total activity,                                                               Protein, activity, μmol/min-                                              Preparation Fraction mg μmol/min mg                                       ______________________________________                                         IC.sub.1 Lysate      1300     7.8    0.006                                        Ni.sup.2+ -NTA 15 2.0 0.13                                                     Phenyl Sepharose 0.40 0.10 0.25                                                Gel Filtration 0.22 0.28 1.3                                                  IIC.sub.2 Lysate 1400 220 0.16                                                  Ni.sup.2+ NTA 63 100 1.6                                                       Mono-Q 57 120 2.1                                                           ______________________________________                                    

Each preparation represents a 10-liter culture. Under the assay conditions defined in FIG. 10, activities were linear with respect to time and protein concentration. However, IC₁ and IIC₂ were not used at saturating concentrations, as defined in FIG. 13B. Thus, the specific activities for the two preparations shown in this table cannot be compared with each other or to the specific activities for IC₁ shown in FIG. 13 and FIG. 14.

The IC₁ fragment accumulated to much lower levels during bacterial culture. It can be purified by a combination of Ni²⁺ -NTA, phenyl-Sepharose, and gel filtration chromatography (FIG. 12 and Table 6). Two distinct peaks of activity were eluted from the phenyl-Sepharose column. The first of these was further purified by gel filtration, where it too displayed an apparent M_(r) of 50,000. The second phenyl-Sepharose peak displayed a similar gel filtration profile but contained more contaminants; this peak was not investigated further.

The inventors first examined adenylyl cyclase activity obtained by mixture of IC₁ and IIC₂ in the absence of any activator (FIG. 13A). Increasing amounts of IIC₂ were added to 0.1 μM IC₁. The highest activity observed (expressed per quantity of IC₁) was 100-fold lower than that obtained in the presence of 50 μM FSK and 500-fold lower than that observed with FSK and activated G_(s)α FIG. 13B). It was not possible to maximize activity by increasing the concentration of IIC₂ in the absence of activators (FIG. 13A). However, activity was maximized with apparent EC₅₀ values for IIC₂ of 190 nM and 15 nM when stimulated by FSK or FSK plus activated G_(s)α, respectively (FIG. 13B). Thus, the apparent affinity of IIC₂ for IC₁ was increased substantially by addition of FSK; an even greater shift was observed when activated G_(s)α was also present.

The inventors also examined the capacity of FSK and G_(s)α to stimulate the adenylyl cyclase activity of the mixed fragments as a function of activator concentration (FIG. 14). In these studies 8 nM IC₁ was mixed with 6.6 μM IIC₂. This is a saturating concentration of IIC₂ in the presence of either FSK or G_(s)α plus FSK. When increasing concentrations of FSK were tested in the presence or absence of 0.5 μM activated G_(s)α, maximal activity was increased by G_(s)α and the EC₅₀ for FSK was lowered by a factor of 100 (FIG. 14A). When increasing concentrations of activated G_(s)α were examined in the presence or absence of 50 μM FSK, maximal activities were similar under the two conditions. However, the inclusion of FSK shifted the Ec₅₀ for activated G_(s)α by more than 100-fold.

Discussion

The inventors have expressed the IC₁ and IIC₂ domains of mammalian adenylyl cyclase separately and reconstituted G_(s)α - and FSK-stimulated adenylyl cyclase activity by their mixture. Neither protein has detectable adenylyl cyclase activity by itself (with or without activators), and the mixture has a very low basal activity in the absence of an activator. However, the activity observed in the presence of FSK and/or activated G_(s)α provides compelling evidence that adenylyl cyclase activity is dependent on the association of the C₁ and C₂ domains of the protein. The stimulated level of activity observed is comparable to that seen with the native, membrane-bound enzyme. Furthermore, the activation produced by FSK and G_(s)α is synergistic. The inventors thus believe that this system provides a valuable tool for definition of mechanisms of regulation of adenylyl cyclase activity. As a first step toward this goal, the inventors provide the model shown in FIG. 15 for analysis of the data presented above.

It is not known if the individual protein fragments studied here exist as dimers, as suggested by their gel filtration profiles. If true, their affinity for homooligomerization is very high, since the gel filtration pattern is unaltered at very low protein concentrations. The inventors also do not know if membrane-bound adenylyl cyclase or the soluble IC₁ IIC₂ construct (Neer et al., 1980; Pfeuffer et al., 1985; Tang et al., 1995) is multimeric. However, the specific activity of IC₁ used in these studies is constant over a broad range of concentrations (200 pM to 100 μM) when assayed in the presence of saturating concentrations of IIC₂ and 50 μM FSK, and the covalent IC₁ IIC₂ construct similarly has a constant specific activity over a similar range of concentrations when activated with either FSK or FSK plus G_(s)α. If the catalytic entity is dependent on the formation of homooligomers, the inventors suggest that such structures are dominant at the protein concentrations used in this study. This justifies consideration of the data presented here in the context of the model shown in FIG. 15.

The data of FIG. 13A provide an estimate of the lower limit for the affinity of IIC₂ for IC₁ in the absence of any activator: K₁ >10 μM (FIG. 15). In the presence of FSK (FIG. 13B), both a higher specific activity and a lower EC₅₀ for IIC₂ are apparent. The expression for fractional activation of IC₁ in the presence of FSK is as follows: ##EQU1##

This neglects the activity due to unregulated complexes of IC₁ and IIC₂, which is insignificant. This derivation also relies on the assumption that K₅ is relatively large--i.e., that most of the IIC₂ in the assay is free and not bound to FSK. At FSK concentrations that are high relative to K₇, Eq. 1 approximates a normal binding isotherm, where the EC₅₀ is equal to K₁ K₇ /[FSK]. From the EC₅₀ of 190 nM (FIG. 13B), K₇ =1 μM. A similar analysis of the FSK activation curve (FIG. 14A) also provides an estimate for K₇ : ##EQU2##

Eq. 2 yields a value for K₇ of 3.7 μM, in reasonable agreement with the value obtained from Eq. 1 (1 μM).

An identical analysis can be done for stimulation of activity by G_(s)α by using Eq. 3 and the EC₅₀ for activated G_(s)α from FIG. 14A. ##EQU3##

This analysis yields a value for K₄ of 0.4 μM. Similarly, the value of K₂ must be sufficiently high to permit this analysis to be meaningful. Attempts are underway to isolate the interactions of the individual fragments with these activators to determine the values of K₂ and K₅.

Qualitatively, it is clear that there is positive cooperativity among the four molecules involved in the formation of active complexes. The inclusion of FSK when titrating IIC₂ shifts the EC₅₀ for IIC₂ from a value of ≧10 μM to 190 nM. Thus, this activator facilitates association of the two adenylyl cyclase fragments (K₆ is less than K₁). The inclusion of activated G_(s)α shifts this EC₅₀ to an even lower value, indicating that G_(s)α and FSK both shift the equilibrium toward association of IC₁ and IIC₂. The binding of these two activators is also positively cooperative with respect to each other. Thus, the presence of activated G_(s)α during the FSK titration lowers the EC₅₀ for FSK dramatically (FIG. 14A), just as the presence of FSK lowers the EC₅₀ for G_(s)α (FIG. 14B). These results are similar to those obtained with the covalently linked IC₁ IIC₂ construct (Dessauer and Gilman, 1996) and the type II enzyme (Feinstein et al., 1991). The inventors conclude that the presence of either activator in the active complex acts to enhance association of the other activator, as well as facilitating the interaction of the C₁ and C₂ domains of adenylyl cyclase.

This model can be confirmed and described explicitly for a single activator if the affinity of the activator for the individual protein domains (K₂ and K₅) can be determined. It is not known which of the two domains, if either, binds FSK or G_(s)α to any significant extent. However, the existence of this system will permit analysis of such interactions.

All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the composition, methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Wallach et al., FEBS Lett., 338:264-266, 1994.

Watson et al., "Molecular Cloning and Characterization of the Type VII Isoform of Mammalian Adenylyl Cyclase Expressed Widely in Mouse Tissures and in S49 Mouse Lymphoma Cells," The Journal of Biological Chemistry, 269(46):28893-28898, November 1994.

Yoshimura and Cooper, Proc. Nat'l Acad. Sci. USA, 89:6716-6720, 1992.

    __________________________________________________________________________     #             SEQUENCE LISTING                                                    - -  - - (1) GENERAL INFORMATION:                                              - -    (iii) NUMBER OF SEQUENCES: 31                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 3978 base - #pairs                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                - - GCCGGCGCCC CAACTCGGCC CGCCGCGCCC CGGCGCCTCG CCGCCCGCCC GC -             #CCGCCCGG     60                                                                  - - CGCCCCGGCC GGCGAGGGGC GCGCCCGCTG CATGGCGCTG GGATGGCGGG GG -             #CGCCGCGC    120                                                                  - - GGCCGAGGCG GCGGCGGAGG CGGAGGCGGC GCGGGCGAGT CTGGGGGCGC CG -             #AGCGGGCG    180                                                                  - - GCGGGGCCGG GCGGCCGGCG CGGGCTGCGG GCGTGCGATG AGGAGTTCGC GT -             #GCCCCGAG    240                                                                  - - CTGGAGGCGC TGTTCCGCGG CTACACGCTG CGGCTGGAGC AGGCGGCGAC GC -             #TGAAGGCG    300                                                                  - - CTGGCCGTGC TCAGCCTGCT GGCGGGCGCG CTGGCCCTGG CCGAGCTGCT GG -             #GCGCGCCG    360                                                                  - - GGGCCCGCGC CCGGCCTGGC TAAGGGCTCG CACCCCGTGC ACTGCGTGCT CT -             #TCCTGGCG    420                                                                  - - CTGCTCGTGG TCACCAACGT CCGCTCGCTG CAGGTGCCCC AGCTGCAGCA GG -             #TCGGCCAG    480                                                                  - - CTCGCGCTGC TCTTCAGCCT CACCTTCGCT CTGCTGTGCT GTCCCTTCGC GC -             #TCGGCGGC    540                                                                  - - CCCGCCGGTG CCCACGCCGG GGCGGCAGCG GTGCCGGCGA CAGCCGATCA GG -             #GAGTCTGG    600                                                                  - - CAGCTCCTTT TGGTCACCTT CGTGTCCTAT GCCCTGCTGC CCGTGCGCAG CC -             #TGCTGGCC    660                                                                  - - ATCGGCTTCG GGCTCGTGGT GGCCGCCTCG CACTTGCTGG TCACGGCTAC GT -             #TGGTCCCC    720                                                                  - - GCCAAGCGCC CACGTCTCTG GAGAACGCTG GGTGCCAACG CTCTGCTCTT CC -             #TCGGTGTG    780                                                                  - - AACGTGTATG GCATCTTCGT GAGGATCCTG GCTGAGCGCG CCCAGAGGAA GG -             #CCTTCCTG    840                                                                  - - CAGGCCCGGA ACTGCATTGA GGACCGGCTG AGGCTGGAGG ATGAGAATGA GA -             #AGCAGGAG    900                                                                  - - CGGCTGCTCA TGAGCCTCCT GCCTCGGAAT GTTGCCATGG AGATGAAGGA GG -             #ACTTCCTG    960                                                                  - - AAGCCCCCTG AGAGGATTTT CCACAAGATT TACATCCAGC GGCATGACAA CG -             #TGAGCATC   1020                                                                  - - CTCTTTGCAG ACATCGTGGG CTTCACAGGC TTGGCGTCAC AGTGCACGGC CC -             #AGGAGCTG   1080                                                                  - - GTGAAACTCC TCAATGAGCT CTTCGGGAAG TTTGACGAGC TGGCCACAGA GA -             #ACCACTGC   1140                                                                  - - CGCCGCATCA AGATCCTGGG AGATTGCTAC TACTGCGTGT CTGGCCTCAC TC -             #AGCCCAAG   1200                                                                  - - ACTGACCACG CCCACTGCTG TGTGGAGATG GGCCTGGACA TGATCGACAC CA -             #TCACGTCC   1260                                                                  - - GTGGCTGAGG CCACTGAGGT GGACTTGAAC ATGCGTGTGG GGCTGCACAC CG -             #GCAGGGTC   1320                                                                  - - CTCTGCGGGG TCCTGGGCCT GCGTAAGTGG CAGTATGATG TGTGGTCCAA CG -             #ACGTGACC   1380                                                                  - - CTGGCCAACG TCATGGAGGC TGCCGGCCTG CCTGGGAAGG TTCACATCAC AA -             #AGACCACC   1440                                                                  - - CTGGCGTGCC TGAATGGTGA CTATGAGGTG GAGCCGGGAC ACGGACACGA GA -             #GGAACAGT   1500                                                                  - - TTTCTGAAAA CTCATAACAT TGAGACCTTT TTTATTGTGC CCTCGCATCG GC -             #GAAAGATA   1560                                                                  - - TTTCCAGGGC TGATTCTCTC AGACATAAAA CCGGCCAAGA GGATGAAGTT CA -             #AGACCGTG   1620                                                                  - - TGCTACCTGC TGGTGCAGCT CATGCACTGC CGGAAGATGT TCAAGGCCGA GA -             #TCCCTTTC   1680                                                                  - - TCCAACGTCA TGACCTGTGA GGATGACGAC AAGCGGAGGG CACTGAGAAC AG -             #CCTCGGAA   1740                                                                  - - AAACTCAGAA ACCGCTCGTC TTTCTCTACA AACGTTGTCC AAACCACCCC CG -             #GCACACGT   1800                                                                  - - GTCAACAGGT ACATCGGCCG CCTCCTGGAA GCCCGCCAGA TGGAGCTGGA GA -             #TGGCAGAC   1860                                                                  - - CTGAACTTCT TCACCCTGAA GTACAAGCAA GCTGAGCGAG AGCGAAAGTA CC -             #ACCAGCTT   1920                                                                  - - CAGGACGAGT ATTTCACCAG CGCCGTGGTT CTGGCTCTCA TTCTGGCCGC CT -             #TATTCGGC   1980                                                                  - - CTTGTCTACC TTCTAATAAT CCCACAGAGT GTGGCTGTCC TGCTCCTGCT GG -             #TGTTCTGC   2040                                                                  - - ATCTGCTTCC TGGTGGCCTG TGTCCTGTAC CTACACATCA CCCGGGTCCA GT -             #GTTTTCCA   2100                                                                  - - GGGTGCCTGA CCATCCAGAT CCGCACCGTC TTGTGCATCT TCATCGTGGT CT -             #TAATCTAC   2160                                                                  - - TCTGTGGCCC AAGGCTGTGT GGTGGGCTGC CTGCCTTGGT CCTGGAGCTC CA -             #GTCCCAAC   2220                                                                  - - GGGTCCCTGG TGGTCCTGTC TTCTGGGGGC CGGGACCCAG TGCTGCCTGT CC -             #CGCCCTGC   2280                                                                  - - GAGTCTGCGC CCCATGCCCT GCTGTGCGGC CTCGTGGGCA CCCTCCCGCT GG -             #CCATATTC   2340                                                                  - - CTGCGGGTCT CCTCCTTGCC AAAAATGATC CTGCTCGCCG TGCTCACCAC CT -             #CCTACATC   2400                                                                  - - CTCGTCCTGG AGCTCAGCGG GTACACGAAG GCCATGGGGG CCGGTGCCAT CT -             #CAGGGCGC   2460                                                                  - - AGCTTCGAGC CGATCATGGC CATCCTGCTA TTCTCGTGCA CGCTGGCCCT GC -             #ACGCCCGG   2520                                                                  - - CAGGTGGATG TCAAGCTGCG GCTGGACTAC CTCTGGGCGG CCCAGGCAGA GG -             #AGGAGCGG   2580                                                                  - - GATGACATGG AGAAAGTGAA GCTGGACAAC AAGAGGATTC TCTTCAACCT CC -             #TGCCAGCC   2640                                                                  - - CACGTTGCCC AGCACTTCCT AATGTCCAAC CCTCGCAACA TGGACCTGTA TT -             #ACCAGTCA   2700                                                                  - - TACTCGCAGG TGGGGGTCAT GTTTGCATCC ATCCCCAACT TCAATGACTT CT -             #ACATCGAG   2760                                                                  - - CTGGATGGCA ACAACATGGG GGTGGAATGT CTACGCCTTC TGAATGAGAT CA -             #TCGCTGAC   2820                                                                  - - TTTGATGAGC TCATGGACAA AGACTTTTAC AAGGACCTAG AGAAGATCAA GA -             #CCATTGGG   2880                                                                  - - AGCACGTACA TGGCTGCTGT GGGGCTGGCG CCCACTGCTG GGACCAAGGC TA -             #AGAAGTGC   2940                                                                  - - ATCTCCTCCC ACCTCAGCAC GTTGGCAGAT TTTGCCATCG AGATGTTTGA TG -             #TCCTGGAT   3000                                                                  - - GAGATCAACT ACCAGTCTTA TAACGACTTT GTGCTCCGTG TTGGCATCAA TG -             #TTGGCCCC   3060                                                                  - - GTGGTGGCTG GAGTGATCGG GGCTCGCAGG CCGCAGTATG ACATTTGGGG GA -             #ACACGGTC   3120                                                                  - - AATGTGGCCA GTCGGATGGA CAGTACCGGC GTCCAGGGCA GGATCCAGGT CA -             #CAGAGGAA   3180                                                                  - - GTTCACCGGC TGCTGCGGCG GGGTTCCTAC CGCTTCGTGT GCCGAGGCAA AG -             #TCAGTGTC   3240                                                                  - - AAGGGCAAGG GTGAGATGCT GACATACTTC CTGGAAGGCA GGACCGATGG AA -             #ATGGCTCC   3300                                                                  - - CAAACCAGGT CCCTGAACTC AGAGCGGAAA ATGTATCCTT TCGGAAGAGC TG -             #GCCTCCAG   3360                                                                  - - ACCAGACTGG CTGCGGGTCA CCCCCCGGTA CCTCCTGCAG CCGGCCTCCC AG -             #TTGGAGCT   3420                                                                  - - GGGCCAGGGG CTCTGCAGGG CTCGGGGCTT GCCCCAGGCC CCCCAGGCCA AC -             #ACCTGCCC   3480                                                                  - - CCTGGAGCCT CTGGGAAGGA GGCTTAGTGG AGCCCATGCC AGCCGCTTGG GG -             #CACAGGGC   3540                                                                  - - ACAAATGCTT GAGGTATTGG GGGTCTTTGG GGCTCCCCCA GGGACCAGCC CA -             #GCCAGCAG   3600                                                                  - - AGCAGGGCTG GGAGCTGGTG ACTGGGCTGG GGAAGGGGCA CAGTCCAGGC AT -             #GACTTGAA   3660                                                                  - - GCAGCTGGGC AGTGAGTCCA GCAGTGAGTC GGGTGAGGGG CGAACCCTGA CC -             #GCACGGAC   3720                                                                  - - ACCGAGGTTT TAGCGGCGAC TGTGTTTGCT TTGTCCTCAC CTCAGTGGCA GA -             #GGGAGGTT   3780                                                                  - - GGCTGGGTCA GCTCCAGGTG TCAGAACGTT CAGGACATTC TCCAGGGCCA TA -             #GGCTACAG   3840                                                                  - - TGAGAGCCTC AGAGGTCTGG CTGGCAGAGT GACTGGGGCG CCCACCTTGT GC -             #CCTGAGAT   3900                                                                  - - GGATCAGCGT CAGCCCAGCA GTGTGGGTCC ATGGGGGCAG CTCCAGCCCT GG -             #GCAGTGGC   3960                                                                  - - CTGCAGAGCC CGGAGTGG             - #                  - #                       - #3978                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1134 amino - #acids                                                (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                - - Met Ala Gly Ala Pro Arg Gly Arg Gly Gly Gl - #y Gly Gly Gly Gly Gly       1               5   - #                10  - #                15                - - Ala Gly Glu Ser Gly Gly Ala Glu Arg Ala Al - #a Gly Pro Gly Gly Arg                   20      - #            25      - #            30                    - - Arg Gly Leu Arg Ala Cys Asp Glu Glu Phe Al - #a Cys Pro Glu Leu Glu               35          - #        40          - #        45                        - - Ala Leu Phe Arg Gly Tyr Thr Leu Arg Leu Gl - #u Gln Ala Ala Thr Leu           50              - #    55              - #    60                            - - Lys Ala Leu Ala Val Leu Ser Leu Leu Ala Gl - #y Ala Leu Ala Leu Ala       65                  - #70                  - #75                  - #80         - - Glu Leu Leu Gly Ala Pro Gly Pro Ala Pro Gl - #y Leu Ala Lys Gly Ser                       85  - #                90  - #                95                - - His Pro Val His Cys Val Leu Phe Leu Ala Le - #u Leu Val Val Thr Asn                   100      - #           105      - #           110                   - - Val Arg Ser Leu Gln Val Pro Gln Leu Gln Gl - #n Val Gly Gln Leu Ala               115          - #       120          - #       125                       - - Leu Leu Phe Ser Leu Thr Phe Ala Leu Leu Cy - #s Cys Pro Phe Ala Leu           130              - #   135              - #   140                           - - Gly Gly Pro Ala Gly Ala His Ala Gly Ala Al - #a Ala Val Pro Ala Thr       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Ala Asp Gln Gly Val Trp Gln Leu Leu Leu Va - #l Thr Phe Val Ser         Tyr                                                                                              165  - #               170  - #               175              - - Ala Leu Leu Pro Val Arg Ser Leu Leu Ala Il - #e Gly Phe Gly Leu Val                   180      - #           185      - #           190                   - - Val Ala Ala Ser His Leu Leu Val Thr Ala Th - #r Leu Val Pro Ala Lys               195          - #       200          - #       205                       - - Arg Pro Arg Leu Trp Arg Thr Leu Gly Ala As - #n Ala Leu Leu Phe Leu           210              - #   215              - #   220                           - - Gly Val Asn Val Tyr Gly Ile Phe Val Arg Il - #e Leu Ala Glu Arg Ala       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Gln Arg Lys Ala Phe Leu Gln Ala Arg Asn Cy - #s Ile Glu Asp Arg         Leu                                                                                              245  - #               250  - #               255              - - Arg Leu Glu Asp Glu Asn Glu Lys Gln Glu Ar - #g Leu Leu Met Ser Leu                   260      - #           265      - #           270                   - - Leu Pro Arg Asn Val Ala Met Glu Met Lys Gl - #u Asp Phe Leu Lys Pro               275          - #       280          - #       285                       - - Pro Glu Arg Ile Phe His Lys Ile Tyr Ile Gl - #n Arg His Asp Asn Val           290              - #   295              - #   300                           - - Ser Ile Leu Phe Ala Asp Ile Val Gly Phe Th - #r Gly Leu Ala Ser Gln       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Cys Thr Ala Gln Glu Leu Val Lys Leu Leu As - #n Glu Leu Phe Gly         Lys                                                                                              325  - #               330  - #               335              - - Phe Asp Glu Leu Ala Thr Glu Asn His Cys Ar - #g Arg Ile Lys Ile Leu                   340      - #           345      - #           350                   - - Gly Asp Cys Tyr Tyr Cys Val Ser Gly Leu Th - #r Gln Pro Lys Thr Asp               355          - #       360          - #       365                       - - His Ala His Cys Cys Val Glu Met Gly Leu As - #p Met Ile Asp Thr Ile           370              - #   375              - #   380                           - - Thr Ser Val Ala Glu Ala Thr Glu Val Asp Le - #u Asn Met Arg Val Gly       385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - Leu His Thr Gly Arg Val Leu Cys Gly Val Le - #u Gly Leu Arg Lys         Trp                                                                                              405  - #               410  - #               415              - - Gln Tyr Asp Val Trp Ser Asn Asp Val Thr Le - #u Ala Asn Val Met Glu                   420      - #           425      - #           430                   - - Ala Ala Gly Leu Pro Gly Lys Val His Ile Th - #r Lys Thr Thr Leu Ala               435          - #       440          - #       445                       - - Cys Leu Asn Gly Asp Tyr Glu Val Glu Pro Gl - #y His Gly His Glu Arg           450              - #   455              - #   460                           - - Asn Ser Phe Leu Lys Thr His Asn Ile Glu Th - #r Phe Phe Ile Val Pro       465                 4 - #70                 4 - #75                 4 -       #80                                                                               - - Ser His Arg Arg Lys Ile Phe Pro Gly Leu Il - #e Leu Ser Asp Ile         Lys                                                                                              485  - #               490  - #               495              - - Pro Ala Lys Arg Met Lys Phe Lys Thr Val Cy - #s Tyr Leu Leu Val Gln                   500      - #           505      - #           510                   - - Leu Met His Cys Arg Lys Met Phe Lys Ala Gl - #u Ile Pro Phe Ser Asn               515          - #       520          - #       525                       - - Val Met Thr Cys Glu Asp Asp Asp Lys Arg Ar - #g Ala Leu Arg Thr Ala           530              - #   535              - #   540                           - - Ser Glu Lys Leu Arg Asn Arg Ser Ser Phe Se - #r Thr Asn Val Val Gln       545                 5 - #50                 5 - #55                 5 -       #60                                                                               - - Thr Thr Pro Gly Thr Arg Val Asn Arg Tyr Il - #e Gly Arg Leu Leu         Glu                                                                                              565  - #               570  - #               575              - - Ala Arg Gln Met Glu Leu Glu Met Ala Asp Le - #u Asn Phe Phe Thr Leu                   580      - #           585      - #           590                   - - Lys Tyr Lys Gln Ala Glu Arg Glu Arg Lys Ty - #r His Gln Leu Gln Asp               595          - #       600          - #       605                       - - Glu Tyr Phe Thr Ser Ala Val Val Leu Ala Le - #u Ile Leu Ala Ala Leu           610              - #   615              - #   620                           - - Phe Gly Leu Val Tyr Leu Leu Ile Ile Pro Gl - #n Ser Val Ala Val Leu       625                 6 - #30                 6 - #35                 6 -       #40                                                                               - - Leu Leu Leu Val Phe Cys Ile Cys Phe Leu Va - #l Ala Cys Val Leu         Tyr                                                                                              645  - #               650  - #               655              - - Leu His Ile Thr Arg Val Gln Cys Phe Pro Gl - #y Cys Leu Thr Ile Gln                   660      - #           665      - #           670                   - - Ile Arg Thr Val Leu Cys Ile Phe Ile Val Va - #l Leu Ile Tyr Ser Val               675          - #       680          - #       685                       - - Ala Gln Gly Cys Val Val Gly Cys Leu Pro Tr - #p Ser Trp Ser Ser Ser           690              - #   695              - #   700                           - - Pro Asn Gly Ser Leu Val Val Leu Ser Ser Gl - #y Gly Arg Asp Pro Val       705                 7 - #10                 7 - #15                 7 -       #20                                                                               - - Leu Pro Val Pro Pro Cys Glu Ser Ala Pro Hi - #s Ala Leu Leu Cys         Gly                                                                                              725  - #               730  - #               735              - - Leu Val Gly Thr Leu Pro Leu Ala Ile Phe Le - #u Arg Val Ser Ser Leu                   740      - #           745      - #           750                   - - Pro Lys Met Ile Leu Leu Ala Val Leu Thr Th - #r Ser Tyr Ile Leu Val               755          - #       760          - #       765                       - - Leu Glu Leu Ser Gly Tyr Thr Lys Ala Met Gl - #y Ala Gly Ala Ile Ser           770              - #   775              - #   780                           - - Gly Arg Ser Phe Glu Pro Ile Met Ala Ile Le - #u Leu Phe Ser Cys Thr       785                 7 - #90                 7 - #95                 8 -       #00                                                                               - - Leu Ala Leu His Ala Arg Gln Val Asp Val Ly - #s Leu Arg Leu Asp         Tyr                                                                                              805  - #               810  - #               815              - - Leu Trp Ala Ala Gln Ala Glu Glu Glu Arg As - #p Asp Met Glu Lys Val                   820      - #           825      - #           830                   - - Lys Leu Asp Asn Lys Arg Ile Leu Phe Asn Le - #u Leu Pro Ala His Val               835          - #       840          - #       845                       - - Ala Gln His Phe Leu Met Ser Asn Pro Arg As - #n Met Asp Leu Tyr Tyr           850              - #   855              - #   860                           - - Gln Ser Tyr Ser Gln Val Gly Val Met Phe Al - #a Ser Ile Pro Asn Phe       865                 8 - #70                 8 - #75                 8 -       #80                                                                               - - Asn Asp Phe Tyr Ile Glu Leu Asp Gly Asn As - #n Met Gly Val Glu         Cys                                                                                              885  - #               890  - #               895              - - Leu Arg Leu Leu Asn Glu Ile Ile Ala Asp Ph - #e Asp Glu Leu Met Asp                   900      - #           905      - #           910                   - - Lys Asp Phe Tyr Lys Asp Leu Glu Lys Ile Ly - #s Thr Ile Gly Ser Thr               915          - #       920          - #       925                       - - Tyr Met Ala Ala Val Gly Leu Ala Pro Thr Al - #a Gly Thr Lys Ala Lys           930              - #   935              - #   940                           - - Lys Cys Ile Ser Ser His Leu Ser Thr Leu Al - #a Asp Phe Ala Ile Glu       945                 9 - #50                 9 - #55                 9 -       #60                                                                               - - Met Phe Asp Val Leu Asp Glu Ile Asn Tyr Gl - #n Ser Tyr Asn Asp         Phe                                                                                              965  - #               970  - #               975              - - Val Leu Arg Val Gly Ile Asn Val Gly Pro Va - #l Val Ala Gly Val Ile                   980      - #           985      - #           990                   - - Gly Ala Arg Arg Pro Gln Tyr Asp Ile Trp Gl - #y Asn Thr Val Asn Val               995          - #       1000          - #      1005                      - - Ala Ser Arg Met Asp Ser Thr Gly Val Gln Gl - #y Arg Ile Gln Val Thr           1010             - #   1015              - #  1020                          - - Glu Glu Val His Arg Leu Leu Arg Arg Gly Se - #r Tyr Arg Phe Val Cys       1025                1030 - #                1035 - #               1040         - - Arg Gly Lys Val Ser Val Lys Gly Lys Gly Gl - #u Met Leu Thr Tyr Phe                       1045 - #               1050  - #              1055              - - Leu Glu Gly Arg Thr Asp Gly Asn Gly Ser Gl - #n Thr Arg Ser Leu Asn                   1060     - #           1065      - #          1070                  - - Ser Glu Arg Lys Met Tyr Pro Phe Gly Arg Al - #a Gly Leu Gln Thr Arg               1075         - #       1080          - #      1085                      - - Leu Ala Ala Gly His Pro Pro Val Pro Pro Al - #a Ala Gly Leu Pro Val           1090             - #   1095              - #  1100                          - - Gly Ala Gly Pro Gly Ala Leu Gln Gly Ser Gl - #y Leu Ala Pro Gly Pro       1105                1110 - #                1115 - #               1120         - - Pro Gly Gln His Leu Pro Pro Gly Ala Ser Gl - #y Lys Glu Ala                               1125 - #               1130                                     - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 4008 base - #pairs                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                - - CCCGGGCAGC GCGCTCTGCG GTCGCCTACC GCCTGCCGCC CCCGCGCCGC CG -              #CGACGTGG     60                                                                  - - CAGGAGGCGA TGCGGCGGCG CCGCTACCTG CGGGACCGCG CCGAGGCGGC GG -             #CGGCAGCG    120                                                                  - - GCGGCGGGAG GCGGAGAGGG GCTGCAGCGG TCCCGGGACT GGCTCTACGA GT -             #CCTACTAC    180                                                                  - - TGCATGAGCC AGCAGCACCC GCTCATCGTC TTCCTGCTGC TCATCGTCAT GG -             #GCGCCTGC    240                                                                  - - CTCGCCCTGC TAGCCGTCTT CTTCGCGCTC GGGCTGGAGG TGGAAGACCA TG -             #TGGCATTT    300                                                                  - - TTAATAACGG TTCCCACTGC CCTGGCCATT TTCTTTGCCA TATTCATTCT TG -             #TCTGCATA    360                                                                  - - GAGTCTGTGT TCAAGAAGCT ACTCCGTGTG TTTTCGCTGG TGATTTGGAT AT -             #GTCTGGTT    420                                                                  - - GCCATGGGAT ACCTGTTCAT GTGCTTCGGA GGGACTGTGT CTGCCTGGGA CC -             #AGGTGTCA    480                                                                  - - TTCTTCCTCT TCATCATCTT TGTGGTATAT ACCATGCTTC CCTTCAACAT GC -             #GAGATGCC    540                                                                  - - ATCATTGCCA GCATCCTCAC ATCTTCATCT CATACGATAG TGCTGAGCGT CT -             #ACCTGTCT    600                                                                  - - GCAACACCAG GGGCCAAGGA GCACCTGTTC TGGCAGATAC TGGCCAATGT GA -             #TCATTTTC    660                                                                  - - ATTTGTGGGA ACTTGGCGGG AGCCTACCAC AAGCACCTCA TGGAGCTTGC CT -             #TGCAGCAA    720                                                                  - - ACCTATCGGG ACACGTGTAA TTGCATCAAG TCCCGGATCA AGCTGGAATT TG -             #AAAAACGG    780                                                                  - - CAGCAGGAAC GGCTCCTGCT CTCCTTGCTG CCAGCTCACA TCGCCATGGA GA -             #TGAAAGCT    840                                                                  - - GAAATCATTC AGAGGCTGCA GGGCCCCAAA GCAGGACAGA TGGAAAACAC AA -             #ACAACTTC    900                                                                  - - CACAATCTGT ATGTCAAACG ACACACCAAC GTGAGCATAT TATACGCTGA CA -             #TTGTTGGC    960                                                                  - - TTCACCCGCC TTGCAAGCGA TTGCTCCCCT GGCGAACTGG TCCACATGCT GA -             #ATGAACTC   1020                                                                  - - TTTGGGAAGT TTGATCAAAT AGCAAAGGAG AATGAATGCA TGAGAATTAA AA -             #TTTTAGGA   1080                                                                  - - GACTGCTATT ACTGTGTTTC CGGGCTCCCT ATATCACTCC CTAACCATGC CA -             #AGAACTGT   1140                                                                  - - GTGAAAATGG GATTGGATAT GTGCGAAGCC ATAAAGAAAG TGAGGGATGC TA -             #CCGGAGTT   1200                                                                  - - GATATCAACA TGCGTGTAGG AGTGCATTCT GGGAACGTTC TCTGTGGTGT GA -             #TTGGTCTC   1260                                                                  - - CAGAAGTGGC AGTATGATGT GTGGTCTCAT GATGTTACTC TGGCAAACCA CA -             #TGGAAGCT   1320                                                                  - - GGAGGAGTCC CTGGGCGTGT TCACATTTCT TCAGTCACTC TGGAGCACTT GA -             #ATGGGGCT   1380                                                                  - - TATAAAGTGG AGGAAGGAGA TGGTGAGATA AGAGACCCAT ATTTAAAGCA GC -             #ACTTGGTG   1440                                                                  - - AAAACCTACT TTGTAATCAA TCCCAAGGGA GAGCGACGGA GTCCTCAGCA TC -             #TCTTCAGA   1500                                                                  - - CCTCGACACA CTCTGGACGG AGCCAAGATG AGAGCATCTG TCCGCATGAC CC -             #GGTACTTG   1560                                                                  - - GAGTCCTGGG GAGCAGCCAA GCCATTCGCA CATCTGCACC ACAGAGATAG CA -             #TGACCACA   1620                                                                  - - GAGAATGGGA AGATTAGTAC CACGGATGTG CCAATGGGTC AACATAATTT TC -             #AAAATCGC   1680                                                                  - - ACCTTAAGAA CTAAGTCACA GAAGAAGAGA TTTGAAGAAG AACTGAATGA AA -             #GGATGATC   1740                                                                  - - CAAGCAATTG ATGGGATCAA TGCACAGAAG CAATGGCTCA AGTCAGAAGA CA -             #TTCAAAGA   1800                                                                  - - ATCTCCCTGC TTTTCTATAA CAAGAATATA GAGAAAGAAT ACCGAGCTAC TG -             #CACTGCCA   1860                                                                  - - GCATTCAAGT ACTACGTGAC CTGTGCCTGC CTCATCTTTC TCTGCATCTT CA -             #TTGTACAG   1920                                                                  - - ATACTTGTAT TGCCAAAAAC GTCCATCCTT GGCTTCTCCT TTGGAGCTGC AT -             #TTCTCTCC   1980                                                                  - - CTCATCTTCA TCCTCTTTGT CTGCTTCGCT GGACAGCTTT TGCAATGCAG CA -             #AAAAGGCC   2040                                                                  - - TCCACCTCTC TCATGTGGCT TTTGAAATCA TCAGGCATCA TCGCCAACCG CC -             #CATGGCCA   2100                                                                  - - CGGATCTCCC TCACAATCGT CACCACGGCT ATCATACTAA CCATGGCTGT GT -             #TCAACATG   2160                                                                  - - TTTTTCCTGA GCAACTCTGA GGAGACAACC CTTCCCACTG CCAATACATC AA -             #ATGCAAAC   2220                                                                  - - GTTTCTGTCC CGGATAACCA GGCGTCGATT CTTCATGCTC GAAACTTGTT TT -             #TCCTCCCG   2280                                                                  - - TACTTCATAT ACAGCTGCAT CCTGGGCTTG ATCTCCTGCT CCGTTTTCCT GA -             #GGGTGAAC   2340                                                                  - - TATGAGTTAA AAATGTTAAT CATGATGGTG GCACTCGTGG GCTACAACAC CA -             #TTCTACTC   2400                                                                  - - CACACCCATG CCCATGTTCT GGATGCGTAC AGCCAGGTCC TGTTTCAGAG AC -             #CAGGCATT   2460                                                                  - - TGGAAAGACC TGAAGACCAT GGGCTCCGTG TCACTCTCCA TATTCTTCAT CA -             #CGCTGCTG   2520                                                                  - - GTTCTGGGCA GACAGAGTGA ATATTACTGT AGGTTAGACT TCTTGTGGAA GA -             #ACAAGTTC   2580                                                                  - - AAAAAAGAGC GGGAGGAGAT AGAAACCATG GAGAACCTAA ATCGAGTGCT GC -             #TGGAGAAC   2640                                                                  - - GTGCTTCCTG CACACGTGGC TGAACACTTC CTGGCCAGGA GCCTGAAAAA TG -             #AGGAGCTG   2700                                                                  - - TACCACCAGT CCTACGACTG TGTCTGTGTC ATGTTTGCCT CCATTCCGGA CT -             #TCAAGGAG   2760                                                                  - - TTCTACACAG AGTCAGATGT GAACAAGGAA GGCTTGGAAT GCCTGCGGCT CC -             #TGAATGAG   2820                                                                  - - ATCATTGCTG ACTTTGATGA TCTGCTTTCT AAGCCAAAGT TCAGTGGTGT TG -             #AAAAGATC   2880                                                                  - - AAGACCATTG GGAGCACATA CATGGCAGCC ACGGGACTGA GTGCCATACC CA -             #GCCAGGAG   2940                                                                  - - CACGCCCAGG AACCTGAGCG TCAGTACATG CACATAGGCA CCATGGTGGA GT -             #TTGCATAT   3000                                                                  - - GCCCTGGTGG GAAAACTGGA TGCCATCAAT AAGCACTCCT TCAACGACTT CA -             #AACTGCGA   3060                                                                  - - GTGGGTATCA ACCATGGGCC TGTAATAGCT GGCGTCATAG GGGCTCAAAA GC -             #CACAGTAT   3120                                                                  - - GACATCTGGG GCAACACTGT CAACGTGGCC AGCAGAATGG ACAGCACCGG GG -             #TCCTGGAC   3180                                                                  - - AAAATACAGG TGACTGAGGA GACAAGCCTC ATCTTGCAGA CGCTTGGCTA CA -             #CGTGTACA   3240                                                                  - - TGTCGAGGTA TCATCAATGT GAAGGGGAAA GGGGACCTGA AGACATATTT TG -             #TAAACACA   3300                                                                  - - GAGATGTCAA GGTCCCTTTC TCAGAGCAAC TTGGCATCCT GAGAAGCTGT CT -             #CTTCCTGA   3360                                                                  - - CAAGAAGAAT GTACTTGCAG GAAGGTACCA CGCACTTTCT GACTGCAACC CT -             #TCCCCTTC   3420                                                                  - - GTCCTGATGT ACGTGCTCTG CCCCATCCTC TGGAGCCCCT GCAGACTAGT TC -             #CTGTGACC   3480                                                                  - - CAGTGACATA CTGTTTGGTG TCTGCGCGTG CCCAGGTTGT CCTGCCACTT GC -             #ACTGTGCT   3540                                                                  - - TGCTCCTAAG CAGGAGGGGA AGGAACCATG TCCTGGAAGG AGAGCATTGG AA -             #GAAGTGAT   3600                                                                  - - GAAGAGGTGA AGTGAACACA CATTCTTAAG GCAATAAAAC CGGGGGGTGT AT -             #ATTATCTT   3660                                                                  - - CTGGTGCATG TTCTTCTCTG GAAAATACGG TAGCTCGTAA CTGCATCCCT AG -             #TCTGATAT   3720                                                                  - - TCAAACACAC AGTATTTGTG AATAAGCTGA TCCCGTCACC CAACATGGAG TC -             #TGTGTTCA   3780                                                                  - - CCTACCCATG TGTCTCATTG CCAGTGGTCG TCCTTGGGGG CTCAGCTGAG AC -             #TCTCAGCT   3840                                                                  - - TCTGTCACCT TGCTGTCCTG TCTTGTGGCA GCAGCACGTT GCCATCCATC AC -             #CAGAATTA   3900                                                                  - - GTCCTCACAG CCTAGGACCA GTTTTGTACC AAACTCATCT GATGTTTTGA TG -             #CCATTTGT   3960                                                                  - - CAAAAGTAAG GTTAATTCAT TAAAAGTTTT ATGTACTTTG AAAAAAAA  - #                   4008                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1090 amino - #acids                                                (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                - - Met Arg Arg Arg Arg Tyr Leu Arg Asp Arg Al - #a Glu Ala Ala Ala Ala       1               5   - #                10  - #                15                - - Ala Ala Ala Gly Gly Gly Glu Gly Leu Gln Ar - #g Ser Arg Asp Trp Leu                   20      - #            25      - #            30                    - - Tyr Glu Ser Tyr Tyr Cys Met Ser Gln Gln Hi - #s Pro Leu Ile Val Phe               35          - #        40          - #        45                        - - Leu Leu Leu Ile Val Met Gly Ala Cys Leu Al - #a Leu Leu Ala Val Phe           50              - #    55              - #    60                            - - Phe Ala Leu Gly Leu Glu Val Glu Asp His Va - #l Ala Phe Leu Ile Thr       65                  - #70                  - #75                  - #80         - - Val Pro Thr Ala Leu Ala Ile Phe Phe Ala Il - #e Phe Ile Leu Val Cys                       85  - #                90  - #                95                - - Ile Glu Ser Val Phe Lys Lys Leu Leu Arg Va - #l Phe Ser Leu Val Ile                   100      - #           105      - #           110                   - - Trp Ile Cys Leu Val Ala Met Gly Tyr Leu Ph - #e Met Cys Phe Gly Gly               115          - #       120          - #       125                       - - Thr Val Ser Ala Trp Asp Gln Val Ser Phe Ph - #e Leu Phe Ile Ile Phe           130              - #   135              - #   140                           - - Val Val Tyr Thr Met Leu Pro Phe Asn Met Ar - #g Asp Ala Ile Ile Ala       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Ser Ile Leu Thr Ser Ser Ser His Thr Ile Va - #l Leu Ser Val Tyr         Leu                                                                                              165  - #               170  - #               175              - - Ser Ala Thr Pro Gly Ala Lys Glu His Leu Ph - #e Trp Gln Ile Leu Ala                   180      - #           185      - #           190                   - - Asn Val Ile Ile Phe Ile Cys Gly Asn Leu Al - #a Gly Ala Tyr His Lys               195          - #       200          - #       205                       - - His Leu Met Glu Leu Ala Leu Gln Gln Thr Ty - #r Arg Asp Thr Cys Asn           210              - #   215              - #   220                           - - Cys Ile Lys Ser Arg Ile Lys Leu Glu Phe Gl - #u Lys Arg Gln Gln Glu       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Arg Leu Leu Leu Ser Leu Leu Pro Ala His Il - #e Ala Met Glu Met         Lys                                                                                              245  - #               250  - #               255              - - Ala Glu Ile Ile Gln Arg Leu Gln Gly Pro Ly - #s Ala Gly Gln Met Glu                   260      - #           265      - #           270                   - - Asn Thr Asn Asn Phe His Asn Leu Tyr Val Ly - #s Arg His Thr Asn Val               275          - #       280          - #       285                       - - Ser Ile Leu Tyr Ala Asp Ile Val Gly Phe Th - #r Arg Leu Ala Ser Asp           290              - #   295              - #   300                           - - Cys Ser Pro Gly Glu Leu Val His Met Leu As - #n Glu Leu Phe Gly Lys       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Phe Asp Gln Ile Ala Lys Glu Asn Glu Cys Me - #t Arg Ile Lys Ile         Leu                                                                                              325  - #               330  - #               335              - - Gly Asp Cys Tyr Tyr Cys Val Ser Gly Leu Pr - #o Ile Ser Leu Pro Asn                   340      - #           345      - #           350                   - - His Ala Lys Asn Cys Val Lys Met Gly Leu As - #p Met Cys Glu Ala Ile               355          - #       360          - #       365                       - - Lys Lys Val Arg Asp Ala Thr Gly Val Asp Il - #e Asn Met Arg Val Gly           370              - #   375              - #   380                           - - Val His Ser Gly Asn Val Leu Cys Gly Val Il - #e Gly Leu Gln Lys Trp       385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - Gln Tyr Asp Val Trp Ser His Asp Val Thr Le - #u Ala Asn His Met         Glu                                                                                              405  - #               410  - #               415              - - Ala Gly Gly Val Pro Gly Arg Val His Ile Se - #r Ser Val Thr Leu Glu                   420      - #           425      - #           430                   - - His Leu Asn Gly Ala Tyr Lys Val Glu Glu Gl - #y Asp Gly Glu Ile Arg               435          - #       440          - #       445                       - - Asp Pro Tyr Leu Lys Gln His Leu Val Lys Th - #r Tyr Phe Val Ile Asn           450              - #   455              - #   460                           - - Pro Lys Gly Glu Arg Arg Ser Pro Gln His Le - #u Phe Arg Pro Arg His       465                 4 - #70                 4 - #75                 4 -       #80                                                                               - - Thr Leu Asp Gly Ala Lys Met Arg Ala Ser Va - #l Arg Met Thr Arg         Tyr                                                                                              485  - #               490  - #               495              - - Leu Glu Ser Trp Gly Ala Ala Lys Pro Phe Al - #a His Leu His His Arg                   500      - #           505      - #           510                   - - Asp Ser Met Thr Thr Glu Asn Gly Lys Ile Se - #r Thr Thr Asp Val Pro               515          - #       520          - #       525                       - - Met Gly Gln His Asn Phe Gln Asn Arg Thr Le - #u Arg Thr Lys Ser Gln           530              - #   535              - #   540                           - - Lys Lys Arg Phe Glu Glu Glu Leu Asn Glu Ar - #g Met Ile Gln Ala Ile       545                 5 - #50                 5 - #55                 5 -       #60                                                                               - - Asp Gly Ile Asn Ala Gln Lys Gln Trp Leu Ly - #s Ser Glu Asp Ile         Gln                                                                                              565  - #               570  - #               575              - - Arg Ile Ser Leu Leu Phe Tyr Asn Lys Asn Il - #e Glu Lys Glu Tyr Arg                   580      - #           585      - #           590                   - - Ala Thr Ala Leu Pro Ala Phe Lys Tyr Tyr Va - #l Thr Cys Ala Cys Leu               595          - #       600          - #       605                       - - Ile Phe Leu Cys Ile Phe Ile Val Gln Ile Le - #u Val Leu Pro Lys Thr           610              - #   615              - #   620                           - - Ser Ile Leu Gly Phe Ser Phe Gly Ala Ala Ph - #e Leu Ser Leu Ile Phe       625                 6 - #30                 6 - #35                 6 -       #40                                                                               - - Ile Leu Phe Val Cys Phe Ala Gly Gln Leu Le - #u Gln Cys Ser Lys         Lys                                                                                              645  - #               650  - #               655              - - Ala Ser Thr Ser Leu Met Trp Leu Leu Lys Se - #r Ser Gly Ile Ile Ala                   660      - #           665      - #           670                   - - Asn Arg Pro Trp Pro Arg Ile Ser Leu Thr Il - #e Val Thr Thr Ala Ile               675          - #       680          - #       685                       - - Ile Leu Thr Met Ala Val Phe Asn Met Phe Ph - #e Leu Ser Asn Ser Glu           690              - #   695              - #   700                           - - Glu Thr Thr Leu Pro Thr Ala Asn Thr Ser As - #n Ala Asn Val Ser Val       705                 7 - #10                 7 - #15                 7 -       #20                                                                               - - Pro Asp Asn Gln Ala Ser Ile Leu His Ala Ar - #g Asn Leu Phe Phe         Leu                                                                                              725  - #               730  - #               735              - - Pro Tyr Phe Ile Tyr Ser Cys Ile Leu Gly Le - #u Ile Ser Cys Ser Val                   740      - #           745      - #           750                   - - Phe Leu Arg Val Asn Tyr Glu Leu Lys Met Le - #u Ile Met Met Val Ala               755          - #       760          - #       765                       - - Leu Val Gly Tyr Asn Thr Ile Leu Leu His Th - #r His Ala His Val Leu           770              - #   775              - #   780                           - - Asp Ala Tyr Ser Gln Val Leu Phe Gln Arg Pr - #o Gly Ile Trp Lys Asp       785                 7 - #90                 7 - #95                 8 -       #00                                                                               - - Leu Lys Thr Met Gly Ser Val Ser Leu Ser Il - #e Phe Phe Ile Thr         Leu                                                                                              805  - #               810  - #               815              - - Leu Val Leu Gly Arg Gln Ser Glu Tyr Tyr Cy - #s Arg Leu Asp Phe Leu                   820      - #           825      - #           830                   - - Trp Lys Asn Lys Phe Lys Lys Glu Arg Glu Gl - #u Ile Glu Thr Met Glu               835          - #       840          - #       845                       - - Asn Leu Asn Arg Val Leu Leu Glu Asn Val Le - #u Pro Ala His Val Ala           850              - #   855              - #   860                           - - Glu His Phe Leu Ala Arg Ser Leu Lys Asn Gl - #u Glu Leu Tyr His Gln       865                 8 - #70                 8 - #75                 8 -       #80                                                                               - - Ser Tyr Asp Cys Val Cys Val Met Phe Ala Se - #r Ile Pro Asp Phe         Lys                                                                                              885  - #               890  - #               895              - - Glu Phe Tyr Thr Glu Ser Asp Val Asn Lys Gl - #u Gly Leu Glu Cys Leu                   900      - #           905      - #           910                   - - Arg Leu Leu Asn Glu Ile Ile Ala Asp Phe As - #p Asp Leu Leu Ser Lys               915          - #       920          - #       925                       - - Pro Lys Phe Ser Gly Val Glu Lys Ile Lys Th - #r Ile Gly Ser Thr Tyr           930              - #   935              - #   940                           - - Met Ala Ala Thr Gly Leu Ser Ala Ile Pro Se - #r Gln Glu His Ala Gln       945                 9 - #50                 9 - #55                 9 -       #60                                                                               - - Glu Pro Glu Arg Gln Tyr Met His Ile Gly Th - #r Met Val Glu Phe         Ala                                                                                              965  - #               970  - #               975              - - Tyr Ala Leu Val Gly Lys Leu Asp Ala Ile As - #n Lys His Ser Phe Asn                   980      - #           985      - #           990                   - - Asp Phe Lys Leu Arg Val Gly Ile Asn His Gl - #y Pro Val Ile Ala Gly               995          - #       1000          - #      1005                      - - Val Ile Gly Ala Gln Lys Pro Gln Tyr Asp Il - #e Trp Gly Asn Thr Val           1010             - #   1015              - #  1020                          - - Asn Val Ala Ser Arg Met Asp Ser Thr Gly Va - #l Leu Asp Lys Ile Gln       1025                1030 - #                1035 - #               1040         - - Val Thr Glu Glu Thr Ser Leu Ile Leu Gln Th - #r Leu Gly Tyr Thr Cys                       1045 - #               1050  - #              1055              - - Thr Cys Arg Gly Ile Ile Asn Val Lys Gly Ly - #s Gly Asp Leu Lys Thr                   1060     - #           1065      - #          1070                  - - Tyr Phe Val Asn Thr Glu Met Ser Arg Ser Le - #u Ser Gln Ser Asn Leu               1075         - #       1080          - #      1085                      - - Ala Ser                                                                       1090                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 4533 base - #pairs                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                - - TGAACCACAG CCCCGCGCCT CTACCGGCCC GACCCGGGGC CTGACCGCAA GG -              #GGACCCGC     60                                                                  - - GCACAGGCTC CGGGCGGAGG AGCTGGCGCG GCGGCACTGC GGGAAGCAGC CT -             #GCGGCTCG    120                                                                  - - CTGAAGCCCG CTTGGCCCGG CTCCGCAGCC CTGGTCCGGA GCGCGCGGCT CC -             #CGAGTTCC    180                                                                  - - CGCACAGTTG CAGATGATCG GTGCCACCTG TGTCCTGGGA ACCCACTAAA CA -             #TTCCAGCT    240                                                                  - - CCAGTTGGAC GCCCTTCACC CACAACCAAG GGGAGCCTCC CCGCCTTCCC AG -             #GTCTTAAC    300                                                                  - - GAAGCCCTGG TGAAGTGGCT TGACCTGGAC TTCTCCCTCC CTCATTAGAA GC -             #AGCCACCA    360                                                                  - - GTCAACATGA CCGAGGACCA GGGCTTCTCG GATCCCGAGT ACTCGGCAGA GT -             #ACTCAGCC    420                                                                  - - GAGTACTCAG TCAGCTTGCC CTCTGACCCC GACCGCGGGG TCGGCCGGAC CC -             #ATGAAATT    480                                                                  - - TCTGTGCGGA ACTCTGGGTC CTGCCTGTGC CTGCCTCGCT TTATGCGGCT GA -             #CCTTCGTG    540                                                                  - - CCTGAGTCCT TGGAGAACCT CTACCAGACC TACTTTAAAA GGCAGCGCCA TG -             #AGACACTG    600                                                                  - - CTGGTGCTGG TGGTCTTTGC GGCCCTCTTT GACTGCTACG TGGTAGTGAT GT -             #GCGCGGTG    660                                                                  - - GTCTTTTCCA GCGACAAGCT GGCGCCCCTC ATGGTGGCAG GCGTCGGTCT GG -             #TGTTGGAC    720                                                                  - - ATCATCCTTT TCGTGCTCTG CAAAAAGGGG CTGCTCCCGG ATCGAGTGAG CC -             #GCAAAGTG    780                                                                  - - GTACCCTACC TGCTGTGGCT GCTCATCACA GCCCAGATCT TCTCCTACCT GG -             #GCCTGAAC    840                                                                  - - TTTTCGCGTG CCCACGCAGC CAGTGACACT GTGGGTTGGC AGGCCTTCTT TG -             #TCTTCTCC    900                                                                  - - TTCTTCATAA CGCTGCCACT CAGCCTCAGC CCCATCGTGA TCATCTCCGT GG -             #TCTCCTGT    960                                                                  - - GTTGTGCATA CGCTTGTCTT GGGGGTCACG GTGGCCCAGC AGCAGCAAGA CG -             #AGCTAGAA   1020                                                                  - - GGGATGCAGC TGCTGAGGGA GATCCTGGCT AACGTCTTCC TCTACCTGTG CG -             #CCATCATC   1080                                                                  - - GTGGGCATCA TGTCCTACTA CATGGCAGAC CGTAAGCACC GAAAGGCCTT CC -             #TGGAGGCC   1140                                                                  - - CGCCAGTCGC TGGAGGTGAA GATGAATCTG GAGGAGCAGA GCCAGCAGCA GG -             #AAAACCTT   1200                                                                  - - ATGCTTTCCA TCCTGCCAAA GCATGTGGCT GACGAGATGT TGAAGGACAT GA -             #AGAAAGAT   1260                                                                  - - GAGAGTCAGA AGGACCAGCA GCAGTTCAAC ACCATGTACA TGTACCGCCA TG -             #AGAATGTC   1320                                                                  - - AGCATCCTGT TTGCAGATAT TGTGGGCTTT ACCCAGCTGT CCTCTGCTTG CA -             #GTGCCCAG   1380                                                                  - - GAGCTCGTGA AGCTACTCAA CGAGCTCTTT GCCCGCTTTG ACAAGCTGGC GG -             #CCAAATAC   1440                                                                  - - CACCAGCTGA GGATCAAGAT CCTAGGCGAC TGTTACTACT GCATCTGCGG CC -             #TGCCTGAC   1500                                                                  - - TACCGGGAGG ACCACGCCGT GTGCTCCATC CTGATGGGGC TTGCCATGGT AG -             #AGGCCATC   1560                                                                  - - TCGTACGTGC GGGAGAAGAC CAAGACCGGA GTGGACATGC GTGTGGGGGT GC -             #ACACAGGC   1620                                                                  - - ACTGTGCTAG GTGGCGTCCT GGGCCAGAAG CGCTGGCAGT ATGATGTATG GT -             #CTACCGAT   1680                                                                  - - GTCACTGTGG CAAACAAGAT GGAGGCTGGC GGCATCCCAG GGCGCGTGCA CA -             #TTTCCCAG   1740                                                                  - - AGCACCATGG ACTGCCTGAA AGGGGAGTTC GATGTCGAAC CTGGTGATGG TG -             #GCAGTCGC   1800                                                                  - - TGCGACTACC TAGATGAGAA GGGCATCGAA ACCTACCTCA TCATTGCCTC CA -             #AGCCAGAG   1860                                                                  - - GTGAAGAAGA CAGCTCAAAA TGGCCTCAAC GGCTCGGCCC TGCCAAACGG AG -             #CACCGGCA   1920                                                                  - - TCCAAGCCCA GCTCCCCTGC CCTTATTGAG ACCAAGGAGC CCAATGGGAG TG -             #CCCATGCC   1980                                                                  - - AGCGGCTCCA CATCAGAGGA GGCTGAAGAA CAGGAGGCCC AGGCTGACAA CC -             #CCTCGTTC   2040                                                                  - - CCCAACCCCC GCCGCAGGCT GCGCCTCCAG GACCTGGCAG ACCGTGTGGT GG -             #ACGCCTCT   2100                                                                  - - GAGGATGAGC ACGAACTGAA CCAGCTTCTT AACGAGGCCC TGCTGGAGCG GG -             #AGTCCGCC   2160                                                                  - - CAGGTGGTAA AGAAGAGAAA CACATTCCTC CTAACGATGA GGTTCATGGA CC -             #CAGAGATG   2220                                                                  - - GAAACACGCT ACTCGGTGGA GAAGGAGAAG CAGAGTGGGG CTGCCTTCAG CT -             #GTTCCTGT   2280                                                                  - - GTGGTCCTTT TCTGCACGGC CATGGTGGAG ATACTTATCG ACCCCTGGTT GA -             #TGACAAAC   2340                                                                  - - TACGTGACCT TCGTGGTTGG AGAGGTTCTG CTCTTGATCC TGACCATCTG TT -             #CGATGGCT   2400                                                                  - - GCCATCTTCC CCAGGGCATT TCCTAAGAAG CTCGTGGCCT TCTCATCTTG GA -             #TTGACCGG   2460                                                                  - - ACCCGCTGGG CCAGAAATAC CTGGGCCATG TTAGCCATCT TCATTCTGGT TA -             #TGGCCAAT   2520                                                                  - - GTTGTGGACA TGCTGAGCTG TCTCCAGTAC TACATGGGAC CTTACAACGT GA -             #CAACCGGG   2580                                                                  - - ATAGAGCTGG ACGGTGGCTG TATGGAGAAC CCCAAGTACT ACAACTATGT TG -             #CTGTGCTG   2640                                                                  - - TCCCTCATCG CCACCATCAT GCTGGTGCAG GTCAGCCACA TGGTGAAGCT GA -             #CACTCATG   2700                                                                  - - CTGCTCGTCA CAGGCGCCGT GACTGCCATC AACCTGTATG CCTGGTGTCC TG -             #TCTTTGAT   2760                                                                  - - GAATACGACC ACAAACGCTT TCAGGAAAAG GACTCTCCTA TGGTGGCCTT AG -             #AGAAGATG   2820                                                                  - - CAGGTACTTT CCACCCCTGG GCTCAATGGC ACTGACAGCA GGCTGCCCCT GG -             #TGCCTTCC   2880                                                                  - - AAGTACTCGA TGACTGTGAT GATGTTCGTT ATGATGCTGA GCTTTTACTA CT -             #TCTCACGC   2940                                                                  - - CACGTGGAGA AACTGGCCCG GACACTGTTC TTGTGGAAGA TTGAGGTCCA TG -             #ACCAGAAA   3000                                                                  - - GAACGTGTGT ACGAGATGCG CCGGTGGAAC GAGGCCTTGG TCACCAACAT GT -             #TGCCAGAG   3060                                                                  - - CATGTTGCAC GCCATTTCCT GGGCTCCAAG AAGAGAGATG AGGAGCTGTA CA -             #GCCAGTCT   3120                                                                  - - TATGACGAGA TTGGAGTCAT GTTTGCCTCC TTGCCCAACT TTGCCGACTT CT -             #ACACTGAG   3180                                                                  - - GAGAGCATCA ATAATGGTGG CATCGAGTGT CTACGCTTCC TCAATGAGAT CA -             #TCTCTGAT   3240                                                                  - - TTTGACTCTC TCCTGGACAA TCCCAAATTC CGGGTCATCA CCAAGATCAA AA -             #CCATCGGC   3300                                                                  - - AGCACCTATA TGGCAGCTTC TGGAGTCACA CCAGATGTCA ACACCAATGG CT -             #TTACAAGC   3360                                                                  - - TCCAGCAAGG AGGAAAAGTC AGACAAGGAG CGCTGGCAGC ACCTGGCTGA CC -             #TGGCAGAC   3420                                                                  - - TTTGCACTAG CCATGAAGGA CACGCTTACA AACATCAACA ACCAGTCATT CA -             #ACAACTTC   3480                                                                  - - ATGCTGCGCA TAGGCATGAA CAAAGGAGGA GTTCTGGCTG GAGTCATTGG AG -             #CCCGGAAG   3540                                                                  - - CCACACTATG ACATCTGGGG GAACACGGTC AATGTGGCCA GCAGGATGGA AT -             #CCACAGGG   3600                                                                  - - GTCATGGGCA ATATCCAGGT GGTAGAAGAG ACACAGGTCA TCCTTCGAGA GT -             #ATGGCTTC   3660                                                                  - - CGCTTTGTGA GGCGAGGACC CATCTTTGTG AAGGGCAAAG GGGAGCTTCT GA -             #CCTTTTTC   3720                                                                  - - TTAAAGGGGC GGGACAGGCC AGCTGCCTTC CCCAATGGCT CCTCTGTTAC AC -             #TGCCCCAC   3780                                                                  - - CAAGTGGTGG ACAACCCCTG AACAGCCCTG GCTCCACAGC GGTCCACACT GG -             #AAGGGAGA   3840                                                                  - - CATTTTGGAA GCAGAGAAAA GCTTTGGGAA AGGGCAAACG ACCGAGTCCC GG -             #GGTTCCCA   3900                                                                  - - ACTGTTGAAG TGCACATTCC CATAGACTTT AGGTTTCAGA TTTCCTCCAG CC -             #TCCCTGTG   3960                                                                  - - TGGCTGTGAG CTCTGAGCGT GGCTATTCCT ACTCCTCAGT GTGCCTGTAG CT -             #TCCCCAAA   4020                                                                  - - GCAGGGGTCT AGGCATAGTA CTGGAGCAGC CTTTCCAGAG CCTTTGTTCC AG -             #CTCAGCCC   4080                                                                  - - TTCACCCCTA GAGAGGCCAC AGCCACTGTG AGCAGGATGC CAGCAGATGT GG -             #GATGAAGC   4140                                                                  - - TGCCTGTGCT GCAGGGTGGG GGCACAGGCT GGGCTCAGGG CCGAGAAGGC TC -             #GGCGCCTG   4200                                                                  - - GCTGCGCCTC CTGTCAGTTA AGTCAATGGC CCTTACCCGA TGTTCCTGAT AA -             #TCTTGAAA   4260                                                                  - - GGTTCTTCTG GAACCCCCGT GTCACCTTAG TCACGAGAGC GAAAAGTGCA AT -             #ATTTCCTT   4320                                                                  - - TCACCTGGTT GGGAAGGGGG GTTATTTCTG AAAGAAAAAA ATATATAAAC AG -             #ATCTTCTA   4380                                                                  - - CATTTATATT TTTAACCTGT TCAAAAAACA ACTTTCCAAT ATTGCCTTGT CT -             #TCTGAGCT   4440                                                                  - - CTTGCTACAG TCGCCTTTGC TACTGCTTTA ACAGAGAATC TGCAGGTGTT GA -             #TAAAGAAC   4500                                                                  - - AGGACTGTTT TATTAAAAGC TTTACTCAAC TTG       - #                  -       #       4533                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1144 amino - #acids                                                (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                - - Met Thr Glu Asp Gln Gly Phe Ser Asp Pro Gl - #u Tyr Ser Ala Glu         Tyr                                                                              1               5   - #                10  - #                15               - - Ser Ala Glu Tyr Ser Val Ser Leu Pro Ser As - #p Pro Asp Arg Gly Val                   20      - #            25      - #            30                    - - Gly Arg Thr His Glu Ile Ser Val Arg Asn Se - #r Gly Ser Cys Leu Cys               35          - #        40          - #        45                        - - Leu Pro Arg Phe Met Arg Leu Thr Phe Val Pr - #o Glu Ser Leu Glu Asn           50              - #    55              - #    60                            - - Leu Tyr Gln Thr Tyr Phe Lys Arg Gln Arg Hi - #s Glu Thr Leu Leu Val       65                  - #70                  - #75                  - #80         - - Leu Val Val Phe Ala Ala Leu Phe Asp Cys Ty - #r Val Val Val Met Cys                       85  - #                90  - #                95                - - Ala Val Val Phe Ser Ser Asp Lys Leu Ala Pr - #o Leu Met Val Ala Gly                   100      - #           105      - #           110                   - - Val Gly Leu Val Leu Asp Ile Ile Leu Phe Va - #l Leu Cys Lys Lys Gly               115          - #       120          - #       125                       - - Leu Leu Pro Asp Arg Val Ser Arg Lys Val Va - #l Pro Tyr Leu Leu Trp           130              - #   135              - #   140                           - - Leu Leu Ile Thr Ala Gln Ile Phe Ser Tyr Le - #u Gly Leu Asn Phe Ser       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Arg Ala His Ala Ala Ser Asp Thr Val Gly Tr - #p Gln Ala Phe Phe         Val                                                                                              165  - #               170  - #               175              - - Phe Ser Phe Phe Ile Thr Leu Pro Leu Ser Le - #u Ser Pro Ile Val Ile                   180      - #           185      - #           190                   - - Ile Ser Val Val Ser Cys Val Val His Thr Le - #u Val Leu Gly Val Thr               195          - #       200          - #       205                       - - Val Ala Gln Gln Gln Gln Asp Glu Leu Glu Gl - #y Met Gln Leu Leu Arg           210              - #   215              - #   220                           - - Glu Ile Leu Ala Asn Val Phe Leu Tyr Leu Cy - #s Ala Ile Ile Val Gly       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Ile Met Ser Tyr Tyr Met Ala Asp Arg Lys Hi - #s Arg Lys Ala Phe         Leu                                                                                              245  - #               250  - #               255              - - Glu Ala Arg Gln Ser Leu Glu Val Lys Met As - #n Leu Glu Glu Gln Ser                   260      - #           265      - #           270                   - - Gln Gln Gln Glu Asn Leu Met Leu Ser Ile Le - #u Pro Lys His Val Ala               275          - #       280          - #       285                       - - Asp Glu Met Leu Lys Asp Met Lys Lys Asp Gl - #u Ser Gln Lys Asp Gln           290              - #   295              - #   300                           - - Gln Gln Phe Asn Thr Met Tyr Met Tyr Arg Hi - #s Glu Asn Val Ser Ile       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Leu Phe Ala Asp Ile Val Gly Phe Thr Gln Le - #u Ser Ser Ala Cys         Ser                                                                                              325  - #               330  - #               335              - - Ala Gln Glu Leu Val Lys Leu Leu Asn Glu Le - #u Phe Ala Arg Phe Asp                   340      - #           345      - #           350                   - - Lys Leu Ala Ala Lys Tyr His Gln Leu Arg Il - #e Lys Ile Leu Gly Asp               355          - #       360          - #       365                       - - Cys Tyr Tyr Cys Ile Cys Gly Leu Pro Asp Ty - #r Arg Glu Asp His Ala           370              - #   375              - #   380                           - - Val Cys Ser Ile Leu Met Gly Leu Ala Met Va - #l Glu Ala Ile Ser Tyr       385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - Val Arg Glu Lys Thr Lys Thr Gly Val Asp Me - #t Arg Val Gly Val         His                                                                                              405  - #               410  - #               415              - - Thr Gly Thr Val Leu Gly Gly Val Leu Gly Gl - #n Lys Arg Trp Gln Tyr                   420      - #           425      - #           430                   - - Asp Val Trp Ser Thr Asp Val Thr Val Ala As - #n Lys Met Glu Ala Gly               435          - #       440          - #       445                       - - Gly Ile Pro Gly Arg Val His Ile Ser Gln Se - #r Thr Met Asp Cys Leu           450              - #   455              - #   460                           - - Lys Gly Glu Phe Asp Val Glu Pro Gly Asp Gl - #y Gly Ser Arg Cys Asp       465                 4 - #70                 4 - #75                 4 -       #80                                                                               - - Tyr Leu Asp Glu Lys Gly Ile Glu Thr Tyr Le - #u Ile Ile Ala Ser         Lys                                                                                              485  - #               490  - #               495              - - Pro Glu Val Lys Lys Thr Ala Gln Asn Gly Le - #u Asn Gly Ser Ala Leu                   500      - #           505      - #           510                   - - Pro Asn Gly Ala Pro Ala Ser Lys Pro Ser Se - #r Pro Ala Leu Ile Glu               515          - #       520          - #       525                       - - Thr Lys Glu Pro Asn Gly Ser Ala His Ala Se - #r Gly Ser Thr Ser Glu           530              - #   535              - #   540                           - - Glu Ala Glu Glu Gln Glu Ala Gln Ala Asp As - #n Pro Ser Phe Pro Asn       545                 5 - #50                 5 - #55                 5 -       #60                                                                               - - Pro Arg Arg Arg Leu Arg Leu Gln Asp Leu Al - #a Asp Arg Val Val         Asp                                                                                              565  - #               570  - #               575              - - Ala Ser Glu Asp Glu His Glu Leu Asn Gln Le - #u Leu Asn Glu Ala Leu                   580      - #           585      - #           590                   - - Leu Glu Arg Glu Ser Ala Gln Val Val Lys Ly - #s Arg Asn Thr Phe Leu               595          - #       600          - #       605                       - - Leu Thr Met Arg Phe Met Asp Pro Glu Met Gl - #u Thr Arg Tyr Ser Val           610              - #   615              - #   620                           - - Glu Lys Glu Lys Gln Ser Gly Ala Ala Phe Se - #r Cys Ser Cys Val Val       625                 6 - #30                 6 - #35                 6 -       #40                                                                               - - Leu Phe Cys Thr Ala Met Val Glu Ile Leu Il - #e Asp Pro Trp Leu         Met                                                                                              645  - #               650  - #               655              - - Thr Asn Tyr Val Thr Phe Val Val Gly Glu Va - #l Leu Leu Leu Ile Leu                   660      - #           665      - #           670                   - - Thr Ile Cys Ser Met Ala Ala Ile Phe Pro Ar - #g Ala Phe Pro Lys Lys               675          - #       680          - #       685                       - - Leu Val Ala Phe Ser Ser Trp Ile Asp Arg Th - #r Arg Trp Ala Arg Asn           690              - #   695              - #   700                           - - Thr Trp Ala Met Leu Ala Ile Phe Ile Leu Va - #l Met Ala Asn Val Val       705                 7 - #10                 7 - #15                 7 -       #20                                                                               - - Asp Met Leu Ser Cys Leu Gln Tyr Tyr Met Gl - #y Pro Tyr Asn Val         Thr                                                                                              725  - #               730  - #               735              - - Thr Gly Ile Glu Leu Asp Gly Gly Cys Met Gl - #u Asn Pro Lys Tyr Tyr                   740      - #           745      - #           750                   - - Asn Tyr Val Ala Val Leu Ser Leu Ile Ala Th - #r Ile Met Leu Val Gln               755          - #       760          - #       765                       - - Val Ser His Met Val Lys Leu Thr Leu Met Le - #u Leu Val Thr Gly Ala           770              - #   775              - #   780                           - - Val Thr Ala Ile Asn Leu Tyr Ala Trp Cys Pr - #o Val Phe Asp Glu Tyr       785                 7 - #90                 7 - #95                 8 -       #00                                                                               - - Asp His Lys Arg Phe Gln Glu Lys Asp Ser Pr - #o Met Val Ala Leu         Glu                                                                                              805  - #               810  - #               815              - - Lys Met Gln Val Leu Ser Thr Pro Gly Leu As - #n Gly Thr Asp Ser Arg                   820      - #           825      - #           830                   - - Leu Pro Leu Val Pro Ser Lys Tyr Ser Met Th - #r Val Met Met Phe Val               835          - #       840          - #       845                       - - Met Met Leu Ser Phe Tyr Tyr Phe Ser Arg Hi - #s Val Glu Lys Leu Ala           850              - #   855              - #   860                           - - Arg Thr Leu Phe Leu Trp Lys Ile Glu Val Hi - #s Asp Gln Lys Glu Arg       865                 8 - #70                 8 - #75                 8 -       #80                                                                               - - Val Tyr Glu Met Arg Arg Trp Asn Glu Ala Le - #u Val Thr Asn Met         Leu                                                                                              885  - #               890  - #               895              - - Pro Glu His Val Ala Arg His Phe Leu Gly Se - #r Lys Lys Arg Asp Glu                   900      - #           905      - #           910                   - - Glu Leu Tyr Ser Gln Ser Tyr Asp Glu Ile Gl - #y Val Met Phe Ala Ser               915          - #       920          - #       925                       - - Leu Pro Asn Phe Ala Asp Phe Tyr Thr Glu Gl - #u Ser Ile Asn Asn Gly           930              - #   935              - #   940                           - - Gly Ile Glu Cys Leu Arg Phe Leu Asn Glu Il - #e Ile Ser Asp Phe Asp       945                 9 - #50                 9 - #55                 9 -       #60                                                                               - - Ser Leu Leu Asp Asn Pro Lys Phe Arg Val Il - #e Thr Lys Ile Lys         Thr                                                                                              965  - #               970  - #               975              - - Ile Gly Ser Thr Tyr Met Ala Ala Ser Gly Va - #l Thr Pro Asp Val Asn                   980      - #           985      - #           990                   - - Thr Asn Gly Phe Thr Ser Ser Ser Lys Glu Gl - #u Lys Ser Asp Lys Glu               995          - #       1000          - #      1005                      - - Arg Trp Gln His Leu Ala Asp Leu Ala Asp Ph - #e Ala Leu Ala Met Lys           1010             - #   1015              - #  1020                          - - Asp Thr Leu Thr Asn Ile Asn Asn Gln Ser Ph - #e Asn Asn Phe Met Leu       1025                1030 - #                1035 - #               1040         - - Arg Ile Gly Met Asn Lys Gly Gly Val Leu Al - #a Gly Val Ile Gly Ala                       1045 - #               1050  - #              1055              - - Arg Lys Pro His Tyr Asp Ile Trp Gly Asn Th - #r Val Asn Val Ala Ser                   1060     - #           1065      - #          1070                  - - Arg Met Glu Ser Thr Gly Val Met Gly Asn Il - #e Gln Val Val Glu Glu               1075         - #       1080          - #      1085                      - - Thr Gln Val Ile Leu Arg Glu Tyr Gly Phe Ar - #g Phe Val Arg Arg Gly           1090             - #   1095              - #  1100                          - - Pro Ile Phe Val Lys Gly Lys Gly Glu Leu Le - #u Thr Phe Phe Leu Lys       1105                1110 - #                1115 - #               1120         - - Gly Arg Asp Arg Pro Ala Ala Phe Pro Asn Gl - #y Ser Ser Val Thr Leu                       1125 - #               1130  - #              1135              - - Pro His Gln Val Val Asp Asn Pro                                                       1140                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 3357 base - #pairs                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                - - CGGGGCAGCC AGCCTACCCA GGTTCCCTCC TGGAGCCTAG CTTTGCTACG AG -              #AACTTCAG     60                                                                  - - GAGCTTAACA CAAAGCAGGC AGGGGCTCCG GGCTGGGGCG GGGGATATCA TG -             #GCCCGCCT    120                                                                  - - CTTCAGTCCC CGGCCTCCTC CCAGCGAAGA TCTCTTCTAC GAGACTTACT AC -             #AGCCTGAG    180                                                                  - - CCAACAGTAT CCACTGCTAA TTCTGCTTCT GGTCATTGTG CTCTGCGCGA TC -             #GTGGCACT    240                                                                  - - GCCCGCCGTC GCCTGGGCCA GCGGCAGGGA GCTGACCTCA GACCCAAGCT TC -             #CTGACAAC    300                                                                  - - TGTGCTGTGT GCTTTGGGTG GCTTCTCCCT GCTTTTGGGG CTCGCTTCCC GG -             #GAGCAGCA    360                                                                  - - ACTGCAGCGA TGGACACGAC CTCTTTCTGG CCTCATATGG GCTGCTTTGC TG -             #GCTCTGGG    420                                                                  - - CTATGGGTTT CTATTCACTG GGGGTGTGGT GAGCGCCTGG GATCAGGTGT CC -             #TTTTTCCT    480                                                                  - - CTTCATCATT TTCACCGTGT ACGCCATGCT GCCCTTGGGC ATGCGGGATG CT -             #GCTGCCGC    540                                                                  - - GGGGGTCATC TCATCCCTCT CACACCTGCT GGTCCTTGGA CTGTACCTTG GG -             #TGGCGGCC    600                                                                  - - TGAGTCGCAG AGGGATCTGC TACCACAGTT GGCAGCGAAT GCGGTGTTGT TC -             #CTGTGTGG    660                                                                  - - GAACGTGGTG GGAGCATACC ACAAGGCACT GATGGAGCGA GCATTGCGCG CC -             #ACGTTCCG    720                                                                  - - GGAGGCTCTT AGCTCCCTGC ATTCACGCCG GAGGTTGGAC ACTGAGAAAA AG -             #CACCAGGA    780                                                                  - - GCACCTCCTC TTGTCTATCC TTCCTGCCTA CCTGGCCCGA GAGATGAAGG CA -             #GAGATCAT    840                                                                  - - GGCTCGGCTG CAGGCTGGAC AGAGCTCACG GCCAGAGAAC ACAAACAACT TT -             #CACAGCCT    900                                                                  - - GTATGTCAAG AGGCACCAAG GAGTGAGTGT GCTGTATGCT GACATCGTGG GC -             #TTCACACG    960                                                                  - - GCTGGCCAGT GAGTGTTCCC CTAAGGAGCT GGTGCTAATG CTCAATGAAC TC -             #TTCGGCAA   1020                                                                  - - ATTCGACCAA ATTGCAAAGG AGCACGAATG CATGCGGATC AAGATCCTGG GA -             #GACTGTTA   1080                                                                  - - CTACTGTGTA TCCGGGCTGC CCCTCTCTCT GCCTGACCAC GCCATCAATT GC -             #GTGCGCAT   1140                                                                  - - GGGACTGGAC ATGTGCCGGG CCATCAGGAA ACTTCGGGTA GCCACCGGTG TG -             #GATATCAA   1200                                                                  - - CATGCGTGTC GGTGTGCACT CAGGCAGCGT CCTCTGTGGA GTCATCGGGC TA -             #CAGAAGTG   1260                                                                  - - GCAGTATGAT GTCTGGTCCC ATGATGTCAC ATTGGCCAAC CATATGGAAG CA -             #GGCGGCGT   1320                                                                  - - TCCAGGACGA GTGCACATCA CAGGGGCCAC GCTGGCCCTG CTGGCAGGAG CT -             #TATGCTGT   1380                                                                  - - GGAGAGAGCA GACATGGAGC ATCGAGACCC ATACCTTCGG GAGCTAGGGG AA -             #CCCACATA   1440                                                                  - - CCTGGTCATT GATCCTTGGG CTGAGGAGGA AGACGAGAAG GGCACCGAGA GA -             #GGATTGCT   1500                                                                  - - GTCTTCTCTA GAGGGGCACA CGATGCGTCC GTCACTACTG ATGACTCGTT AT -             #CTGGAGTC   1560                                                                  - - TTGGGGTGCA GCCAAGCCTT TCGCCCACCT AAGCCACGTC GACAGTCCTG CA -             #TCCACATC   1620                                                                  - - CACTCCACTC CCGGAGAAAG CCTTCAGCCC CCAGTGGAGC CTGGACCGGA GT -             #CGCACCCC   1680                                                                  - - CCGCGGGCTA CACGATGAAC TGGACACTGG AGATGCCAAG TTCTTCCAGG TC -             #ATCGAACA   1740                                                                  - - ACTCAACTCT CAAAAACAGT GGAAACAGTC GAAGGACTTC AACCTCCTGA CG -             #CTCTACTT   1800                                                                  - - CAGAGAGAAG GAGATGGAGA AACAGTATCG GCTGTCTGCG CTCCCCGCCT TC -             #AAATACTA   1860                                                                  - - CGCAGCCTGC ACCTTCCTGG TTTTTCTGTC CAACTTCACA ATCCAAATGC TG -             #GTGACAAC   1920                                                                  - - CAGGCCCCCA GCTCTGGCCA CCACCTACAG CATCACCTTC CTTCTCTTCC TT -             #CTCCTCCT   1980                                                                  - - CTTCGTCTGC TTCTCAGAGC ACCTGACGAA GTGCGTCCAG AAAGGCCCCA AG -             #ATGTTGCA   2040                                                                  - - CTGGCTGCCT GCGCTGTCTG TCCTGGTGGC CACACGGCCG GGATTGCGAG TA -             #GCCCTGGG   2100                                                                  - - CACAGCCACC ATCCTCCTGG TGTTCACTAT GGCCGTCGTC AGCCTGCTCT TC -             #TTACCAGT   2160                                                                  - - GTCGTCAGAC TGCCCTTTCC TGGCTCCCAA TGTGTCGTCA GTGGCTTTTA AT -             #ACTTCCTG   2220                                                                  - - GGAGCTGCCA GCATCCCTGC CTCTCATCAG CATCCCATAC TCCATGCATT GT -             #TGCGTGCT   2280                                                                  - - GGGTTTCCTT TCCTGCTCCC TTTTTCTGCA CATGAGCTTC GAACTGAAGC TG -             #CTTCTGCT   2340                                                                  - - TCTGCTGTGG CTGGTGGCAT CTTGTTCCCT ATTTCTGCAC TCCCACGCCT GG -             #CTGTCCGA   2400                                                                  - - CTGCCTCATT GCCCGCCTTT ATCAAGGCTC ATTGGGCTCC AGGCCGGGGG TA -             #CTGAAGGA   2460                                                                  - - ACCGAAACTG ATGGGAGCTA TCTACTTCTT CATCTTCTTC TTCACACTCC TC -             #GTCCTGGC   2520                                                                  - - TCGGCAGAAT GAGTATTACT GTCGCCTGGA CTTCCTATGG AAAAAGAAAC TG -             #AGGCAGGA   2580                                                                  - - GCGAGAGGAA ACTGAGACAA TGGAGAATGT ACTCCCTGCA CACGTGGCGC CC -             #CAGCTCAT   2640                                                                  - - CGGCCAGAAC CGGCGCAATG AGGACCTCTA CCATCAGTCG TACGAATGTG TT -             #TGTGTCCT   2700                                                                  - - CTTTGCATCC ATCCCAGACT TTAAGGAATT CTACTCGGAA TCCAACATCA AC -             #CATGAGGG   2760                                                                  - - GCTAGAGTGT CTGCGGCTGC TCAATGAGAT CATTGCCGAC TTTGATGAGC TG -             #CTCTCCAA   2820                                                                  - - GCCAAAGTTC AGTGGAGTAG AGAAGATCAA AACTATCGGC AGCACCTACA TG -             #GCGGCCAC   2880                                                                  - - AGGCCTAAAT GCCACCCCTG GGCAGGACAC ACAACAGGAC GCTGAGAGAA GC -             #TGCAGCCA   2940                                                                  - - TCTGGGCACC ATGGTGGAAT TTGCAGTGGC CCTGGGGTCT AAGCTGGGTG TC -             #ATCAATAA   3000                                                                  - - GCACTCGTTC AACAACTTCC GCCTGCGTGT GGGGTTAAAC CATGGACCAG TC -             #GTAGCAGG   3060                                                                  - - GGTGATTGGG GCACAGAAGC CACAATATGA CATCTGGGGG AATACAGTGA AC -             #GTGGCCAG   3120                                                                  - - CCGCATGGAG AGCACAGGAG TTCTCGGCAA GATCCAAGTG ACCGAGGAGA CA -             #GCTAGGGC   3180                                                                  - - CCTGCAATCC CTGGGTTACA CATGCTACAG CCGAGGTGTC ATCAAGGTCA AG -             #GGCAAAGG   3240                                                                  - - GCAACTCTGT ACCTACTTCC TGAACACAGA CCTGACACGA ACCGGATCTC CC -             #TCAGCATC   3300                                                                  - - CTAGACACCT GAGCTCCCCT TCTCCAAGAT CCTCAATAAA ATGTCTCCAG GC - #ATCTG           3357                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1064 amino - #acids                                                (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                - - Met Ala Arg Leu Phe Ser Pro Arg Pro Pro Pr - #o Ser Glu Asp Leu Phe       1               5   - #                10  - #                15                - - Tyr Glu Thr Tyr Tyr Ser Leu Ser Gln Gln Ty - #r Pro Leu Leu Ile Leu                   20      - #            25      - #            30                    - - Leu Leu Val Ile Val Leu Cys Ala Ile Val Al - #a Leu Pro Ala Val Ala               35          - #        40          - #        45                        - - Trp Ala Ser Gly Arg Glu Leu Thr Ser Asp Pr - #o Ser Phe Leu Thr Thr           50              - #    55              - #    60                            - - Val Leu Cys Ala Leu Gly Gly Phe Ser Leu Le - #u Leu Gly Leu Ala Ser       65                  - #70                  - #75                  - #80         - - Arg Glu Gln Gln Leu Gln Arg Trp Thr Arg Pr - #o Leu Ser Gly Leu Ile                       85  - #                90  - #                95                - - Trp Ala Ala Leu Leu Ala Leu Gly Tyr Gly Ph - #e Leu Phe Thr Gly Gly                   100      - #           105      - #           110                   - - Val Val Ser Ala Trp Asp Gln Val Ser Phe Ph - #e Leu Phe Ile Ile Phe               115          - #       120          - #       125                       - - Thr Val Tyr Ala Met Leu Pro Leu Gly Met Ar - #g Asp Ala Ala Ala Ala           130              - #   135              - #   140                           - - Gly Val Ile Ser Ser Leu Ser His Leu Leu Va - #l Leu Gly Leu Tyr Leu       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Gly Trp Arg Pro Glu Ser Gln Arg Asp Leu Le - #u Pro Gln Leu Ala         Ala                                                                                              165  - #               170  - #               175              - - Asn Ala Val Leu Phe Leu Cys Gly Asn Val Va - #l Gly Ala Tyr His Lys                   180      - #           185      - #           190                   - - Ala Leu Met Glu Arg Ala Leu Arg Ala Thr Ph - #e Arg Glu Ala Leu Ser               195          - #       200          - #       205                       - - Ser Leu His Ser Arg Arg Arg Leu Asp Thr Gl - #u Lys Lys His Gln Glu           210              - #   215              - #   220                           - - His Leu Leu Leu Ser Ile Leu Pro Ala Tyr Le - #u Ala Arg Glu Met Lys       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Ala Glu Ile Met Ala Arg Leu Gln Ala Gly Gl - #n Ser Ser Arg Pro         Glu                                                                                              245  - #               250  - #               255              - - Asn Thr Asn Asn Phe His Ser Leu Tyr Val Ly - #s Arg His Gln Gly Val                   260      - #           265      - #           270                   - - Ser Val Leu Tyr Ala Asp Ile Val Gly Phe Th - #r Arg Leu Ala Ser Glu               275          - #       280          - #       285                       - - Cys Ser Pro Lys Glu Leu Val Leu Met Leu As - #n Glu Leu Phe Gly Lys           290              - #   295              - #   300                           - - Phe Asp Gln Ile Ala Lys Glu His Glu Cys Me - #t Arg Ile Lys Ile Leu       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Gly Asp Cys Tyr Tyr Cys Val Ser Gly Leu Pr - #o Leu Ser Leu Pro         Asp                                                                                              325  - #               330  - #               335              - - His Ala Ile Asn Cys Val Arg Met Gly Leu As - #p Met Cys Arg Ala Ile                   340      - #           345      - #           350                   - - Arg Lys Leu Arg Val Ala Thr Gly Val Asp Il - #e Asn Met Arg Val Gly               355          - #       360          - #       365                       - - Val His Ser Gly Ser Val Leu Cys Gly Val Il - #e Gly Leu Gln Lys Trp           370              - #   375              - #   380                           - - Gln Tyr Asp Val Trp Ser His Asp Val Thr Le - #u Ala Asn His Met Glu       385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - Ala Gly Gly Val Pro Gly Arg Val His Ile Th - #r Gly Ala Thr Leu         Ala                                                                                              405  - #               410  - #               415              - - Leu Leu Ala Gly Ala Tyr Ala Val Glu Arg Al - #a Asp Met Glu His Arg                   420      - #           425      - #           430                   - - Asp Pro Tyr Leu Arg Glu Leu Gly Glu Pro Th - #r Tyr Leu Val Ile Asp               435          - #       440          - #       445                       - - Pro Trp Ala Glu Glu Glu Asp Glu Lys Gly Th - #r Glu Arg Gly Leu Leu           450              - #   455              - #   460                           - - Ser Ser Leu Glu Gly His Thr Met Arg Pro Se - #r Leu Leu Met Thr Arg       465                 4 - #70                 4 - #75                 4 -       #80                                                                               - - Tyr Leu Glu Ser Trp Gly Ala Ala Lys Pro Ph - #e Ala His Leu Ser         His                                                                                              485  - #               490  - #               495              - - Val Asp Ser Pro Ala Ser Thr Ser Thr Pro Le - #u Pro Glu Lys Ala Phe                   500      - #           505      - #           510                   - - Ser Pro Gln Trp Ser Leu Asp Arg Ser Arg Th - #r Pro Arg Gly Leu His               515          - #       520          - #       525                       - - Asp Glu Leu Asp Thr Gly Asp Ala Lys Phe Ph - #e Gln Val Ile Glu Gln           530              - #   535              - #   540                           - - Leu Asn Ser Gln Lys Gln Trp Lys Gln Ser Ly - #s Asp Phe Asn Leu Leu       545                 5 - #50                 5 - #55                 5 -       #60                                                                               - - Thr Leu Tyr Phe Arg Glu Lys Glu Met Glu Ly - #s Gln Tyr Arg Leu         Ser                                                                                              565  - #               570  - #               575              - - Ala Leu Pro Ala Phe Lys Tyr Tyr Ala Ala Cy - #s Thr Phe Leu Val Phe                   580      - #           585      - #           590                   - - Leu Ser Asn Phe Thr Ile Gln Met Leu Val Th - #r Thr Arg Pro Pro Ala               595          - #       600          - #       605                       - - Leu Ala Thr Thr Tyr Ser Ile Thr Phe Leu Le - #u Phe Leu Leu Leu Leu           610              - #   615              - #   620                           - - Phe Val Cys Phe Ser Glu His Leu Thr Lys Cy - #s Val Gln Lys Gly Pro       625                 6 - #30                 6 - #35                 6 -       #40                                                                               - - Lys Met Leu His Trp Leu Pro Ala Leu Ser Va - #l Leu Val Ala Thr         Arg                                                                                              645  - #               650  - #               655              - - Pro Gly Leu Arg Val Ala Leu Gly Thr Ala Th - #r Ile Leu Leu Val Phe                   660      - #           665      - #           670                   - - Thr Met Ala Val Val Ser Leu Leu Phe Leu Pr - #o Val Ser Ser Asp Cys               675          - #       680          - #       685                       - - Pro Phe Leu Ala Pro Asn Val Ser Ser Val Al - #a Phe Asn Thr Ser Trp           690              - #   695              - #   700                           - - Glu Leu Pro Ala Ser Leu Pro Leu Ile Ser Il - #e Pro Tyr Ser Met His       705                 7 - #10                 7 - #15                 7 -       #20                                                                               - - Cys Cys Val Leu Gly Phe Leu Ser Cys Ser Le - #u Phe Leu His Met         Ser                                                                                              725  - #               730  - #               735              - - Phe Glu Leu Lys Leu Leu Leu Leu Leu Leu Tr - #p Leu Val Ala Ser Cys                   740      - #           745      - #           750                   - - Ser Leu Phe Leu His Ser His Ala Trp Leu Se - #r Asp Cys Leu Ile Ala               755          - #       760          - #       765                       - - Arg Leu Tyr Gln Gly Ser Leu Gly Ser Arg Pr - #o Gly Val Leu Lys Glu           770              - #   775              - #   780                           - - Pro Lys Leu Met Gly Ala Ile Tyr Phe Phe Il - #e Phe Phe Phe Thr Leu       785                 7 - #90                 7 - #95                 8 -       #00                                                                               - - Leu Val Leu Ala Arg Gln Asn Glu Tyr Tyr Cy - #s Arg Leu Asp Phe         Leu                                                                                              805  - #               810  - #               815              - - Trp Lys Lys Lys Leu Arg Gln Glu Arg Glu Gl - #u Thr Glu Thr Met Glu                   820      - #           825      - #           830                   - - Asn Val Leu Pro Ala His Val Ala Pro Gln Le - #u Ile Gly Gln Asn Arg               835          - #       840          - #       845                       - - Arg Asn Glu Asp Leu Tyr His Gln Ser Tyr Gl - #u Cys Val Cys Val Leu           850              - #   855              - #   860                           - - Phe Ala Ser Ile Pro Asp Phe Lys Glu Phe Ty - #r Ser Glu Ser Asn Ile       865                 8 - #70                 8 - #75                 8 -       #80                                                                               - - Asn His Glu Gly Leu Glu Cys Leu Arg Leu Le - #u Asn Glu Ile Ile         Ala                                                                                              885  - #               890  - #               895              - - Asp Phe Asp Glu Leu Leu Ser Lys Pro Lys Ph - #e Ser Gly Val Glu Lys                   900      - #           905      - #           910                   - - Ile Lys Thr Ile Gly Ser Thr Tyr Met Ala Al - #a Thr Gly Leu Asn Ala               915          - #       920          - #       925                       - - Thr Pro Gly Gln Asp Thr Gln Gln Asp Ala Gl - #u Arg Ser Cys Ser His           930              - #   935              - #   940                           - - Leu Gly Thr Met Val Glu Phe Ala Val Ala Le - #u Gly Ser Lys Leu Gly       945                 9 - #50                 9 - #55                 9 -       #60                                                                               - - Val Ile Asn Lys His Ser Phe Asn Asn Phe Ar - #g Leu Arg Val Gly         Leu                                                                                              965  - #               970  - #               975              - - Asn His Gly Pro Val Val Ala Gly Val Ile Gl - #y Ala Gln Lys Pro Gln                   980      - #           985      - #           990                   - - Tyr Asp Ile Trp Gly Asn Thr Val Asn Val Al - #a Ser Arg Met Glu Ser               995          - #       1000          - #      1005                      - - Thr Gly Val Leu Gly Lys Ile Gln Val Thr Gl - #u Glu Thr Ala Arg Ala           1010             - #   1015              - #  1020                          - - Leu Gln Ser Leu Gly Tyr Thr Cys Tyr Ser Ar - #g Gly Val Ile Lys Val       1025                1030 - #                1035 - #               1040         - - Lys Gly Lys Gly Gln Leu Cys Thr Tyr Phe Le - #u Asn Thr Asp Leu Thr                       1045 - #               1050  - #              1055              - - Arg Thr Gly Ser Pro Ser Ala Ser                                                       1060                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 3924 base - #pairs                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                - - GTCGAGGAAA AGGCCGAGCG GCCGAGGAGC TGGAGCCCGG AACTGGCACC GT -              #CGAGGATG     60                                                                  - - GAGACGGGTC CGAGGATGGA GGCAGTTCCG TGGCGTCAGG CTCTGGGACC GG -             #CACGGTGC    120                                                                  - - TGTCGTTGGG CGCCTGCTGC CTGGCCTTGC TGCAGATATT CCGCTCTAAG AA -             #GTTCCCGT    180                                                                  - - CGGACAAACT GGAGCGTCTG TACCAGCGCT ATTTCTTCCG CCTGAACCAG AG -             #CAGCCTCA    240                                                                  - - CCATGCTCAT GGCCGTGCTG GTGCTCGTGT GCCTGGTCAT GCTCGCTTTC CA -             #CGCGGCAC    300                                                                  - - GGCCCCCGCT CCAGGTAGTC TACCTGGCCG TGTTGGCAGC TGCTGTGGGC GT -             #GATCCTTA    360                                                                  - - TCATGGCTGT GCTCTGCAAC CGTGCAGCCT TCCACCAGGA CCACATGGGC CT -             #GGCCTGCT    420                                                                  - - ATGCGCTCAT TGCAGTGGTG CTGGCCGTCC AGGTAGTGGG CCTGTTGCTG CC -             #ACAGCCAC    480                                                                  - - GCAGCGCCTC CGAGGGCATC TGGTGGACCG TGTTCTTCAT CTATACCATC TA -             #CACCCTGC    540                                                                  - - TGCCTGTGCG CATGAGGGCT GCGGTGCTCA GCGGGGTGCT TCTGTCGGCT CT -             #CCACTTGG    600                                                                  - - CCATCTCTCT GCACACCAAC GCCCAGGACC AGTTTCTGCT GAAACAGCTT GT -             #CTCCAACG    660                                                                  - - TCCTCATCTT CTCTTGCACC AACATCGTGG GTGTGTGCAC TCACTACCCA GC -             #CGAGGTCT    720                                                                  - - CCCAGAGACA AGCCTTCCAG GAGACCCGGG AGTGCATCCA AGCTCGGCTC CA -             #CTCACAGC    780                                                                  - - GGGAGAACCA GCAACAGGAG CGTCTCCTGC TGTCTGTCCT TCCCCGTCAT GT -             #TGCCATGG    840                                                                  - - AGATGAAAGC AGACATCAAC GCCAAACAGG AGGATATGAT GTTCCACAAG AT -             #TTACATCC    900                                                                  - - AGAAACATGA CAATGTGAGC ATCCTGTTTG CTGACATCGA AGGCTTCACT AG -             #CCTGGCAT    960                                                                  - - CCCAGTGTAC TGCCCAAGAA CTGGTCATGA CCCTCAACGA GCTCTTCGCC CG -             #CTTTGACA   1020                                                                  - - AGTTGGCTGC GGAGAATCAC TGCTTACGGA TTAAGATCCT CGGGGATTGT TA -             #CTACTGTG   1080                                                                  - - TCTCGGGGCT GCCTGAAGCC AGAGCTGACC ACGCCCACTG CTGCGTAGAG AT -             #GGGAATGG   1140                                                                  - - ACATGATCGA GGCCATCTCG TCGGTCCGGG AGGTGACAGG GGTGAACGTG AA -             #CATGCGTG   1200                                                                  - - TGGGAATTCA CAGCGGGAGA GTACACTGCG GTGTCCTTGG CCTCAGAAAG TG -             #GCAATTCG   1260                                                                  - - ACGTGTGGTC TAACGATGTC ACACTGGCCA ACCACATGGA AGCTGGCGGC AA -             #GGCAGGCC   1320                                                                  - - GCATCCACAT CACCAAGGCC ACACTCAACT ACCTGAACGG GGACTATGAG GT -             #GGAGCCAG   1380                                                                  - - GCTGTGGTGG TGAGCGCAAT GCCTACCTCA AGGAGCACAG CATCGAGACC TT -             #CCTCATCC   1440                                                                  - - TGCGCTGTAC CCAGAAGCGG AAAGAAGAGA AGGCCATGAT CGCCAAGATG AA -             #CCGCCAGA   1500                                                                  - - GAACCAACTC CATTGGACAC AATCCGCCTC ACTGGGGAGC TGAGCGCCCC TT -             #CTACAACC   1560                                                                  - - ACTTGGGTGG CAACCAGGTG TCCAAGGAAA TGAAGAGGAT GGGCTTTGAG GA -             #CCCCAAGG   1620                                                                  - - ACAAGAATGC CCAGGAAAGT GCCAACCCTG AGGATGAAGT GGACGAGTTT CT -             #GGGTCGAG   1680                                                                  - - CCATCGATGC CAGGAGTATT GACAGACTGC GATCCGAACA CGTCCGAAAG TT -             #CCTCTTGA   1740                                                                  - - CCTTTAGGGA GCCCGACTTA GAGAAGAAGT ACTCCAAGCA GGTGGATGAC CG -             #ATTTGGTG   1800                                                                  - - CCTATGTGGC CTGTGCCTCG CTTGTTTTCC TCTTCATCTG CTTTGTCCAG AT -             #CACCATTG   1860                                                                  - - TGCCCCACTC CCTGTTCATG CTGAGCTTCT ACCTGTCGTG TTTCCTGCTA CT -             #GGCCTTGG   1920                                                                  - - TGGTGTTTAT ATCTGTGATC TACGCCTGTG TGAAGCTCTT CCCTACTCCC CT -             #GCAGACAC   1980                                                                  - - TCTCCAGGAA GATAGTGCGA TCCAAGAAGA ACAGCACCCT GGTCGGGGTG TT -             #CACCATCA   2040                                                                  - - CCCTGGTGTT CCTCTCGGCT TTTGTCAACA TGTTCATGTG CAACTCTAAG AA -             #CCTGGTGG   2100                                                                  - - GTTGCCTGGC AGAGGAGCAC AACATCACGG TGAACCAGGT GAACGCATGT CA -             #TGTGATGG   2160                                                                  - - AGTCGGCCTT CAACTACAGC CTGGGCGACG AGCAGGGCTT CTGTGGCAGC CC -             #CCAGTCCA   2220                                                                  - - ACTGCAACTT CCCAGAGTAC TTCACCTACA GCGTGCTGCT CAGCCTGCTG GC -             #CTGCTCCG   2280                                                                  - - TGTTCCTGCA GATCAGCTGC ATCGGGAAGC TGGTGCTCAT GCTGGCCATC GA -             #GCTCATCT   2340                                                                  - - ACGTGCTCAT CGTAGAGGTG CCCGGTGTCA CACTCTTTGA CAACGCTGAC CT -             #TCTGGTCA   2400                                                                  - - CCGCCAATGC CATAGACTTC AGCAACAACG GGACCTCCCA GTGCCCTGAG CA -             #TGCGACCA   2460                                                                  - - AGGTGGCGCT GAAGGTGGTG ACGCCCATCA TCATCTCTGT CTTCGTGCTG GC -             #TCTGTATC   2520                                                                  - - TGCATGCTCA GCAGGTGGAA TCCACTGCTC GCCTTGACTT CCTCTGGAAA CT -             #GCAGGCCA   2580                                                                  - - CAGAAGAGAA GGAGGAGATG GAGGAGCTGC AGGCCTACAA CCGGCGCTTG CT -             #GCACAACA   2640                                                                  - - TTCTGCCCAA GGACGTGGCT GCCCACTTCC TGGCCCGGGA GCGGCGCAAT GA -             #TGAACTGT   2700                                                                  - - ACTACCAATC CTGCGAGTGC GTGGCTGTCA TGTTTGCCTC CATCGCCAAC TT -             #CTCCGAAT   2760                                                                  - - TCTACGTGGA GCTAGAGGCC AACAATGAGG GCGTTGAATG CCTACGGCTG CT -             #CAATGAGA   2820                                                                  - - TCATCGCAGA CTTTGATGAG ATCATCAGTG AGGATCGGTT CAGGCAGCTG GA -             #AAAGATCA   2880                                                                  - - AGACCATAGG TAGCACCTAC ATGGCTGCCT CTGGCCTCAA CGACTCCACC TA -             #TGACAAGG   2940                                                                  - - CAGGCAAGAC CCACATCAAG GCTCTTGCAG ACTTCGCCAT GAAGCTGATG GA -             #CCAAATGA   3000                                                                  - - AGTACATCAA TGAGCACTCC TTCAACAACT TCCAGATGAA GATCGGGCTT AA -             #CATTGGAC   3060                                                                  - - CTGTAGTGGC TGGGGTCATT GGGGCTCGCA AGCCTCAGTA TGACATCTGG GG -             #CAATACAG   3120                                                                  - - TAAATGTGGC CAGCCGTATG GACAGCACTG GGGTGCCTGA CCGCATCCAG GT -             #TACTACAG   3180                                                                  - - ATATGTACCA GGTGCTGGCC GCCAACACAT ACCAGCTGGA GTGCCGGGGT GT -             #GGTCAAGG   3240                                                                  - - TCAAGGGCAA GGGTGAGATG ATGACCTACT TCCTCAATGG AGGGCCTCCC CT -             #CAGTTAGC   3300                                                                  - - AGATACCAGC AGCATCCTGG CCTCCAGAGG ATTGAACAGC TTCTCTGTGC AC -             #CATGGGCA   3360                                                                  - - GATAGGGCCT GTGCCCAGGC TCACAGCTGT GCTGGTAAAA TTTCTACTTG GA -             #CTCAGAAG   3420                                                                  - - CAACTTCTGC CTTTGCTGGT GGGCATGCAT TTTGGCCTAG GCCAGGGTGC CA -             #GCGTCCTG   3480                                                                  - - CGAGCACCGA GCTGACCAAA GATGTTGCCC TTGGCAGAAG ACTGTAGACT CG -             #AGCTGACC   3540                                                                  - - CTTGAGGTTC TGACAAGTGC TGCTACTGCA CGGTGGATGT GCTACCGGAG CA -             #CAGCCTGG   3600                                                                  - - CACAGAGTGG CTGGCCTGAG GGGAGGCCTT GGTTATGAAG GGGAGGTAGG CA -             #AGCTTGAA   3660                                                                  - - GCTGGCCTGG GGGATGTCCA TGGACCTCAT GGGTCTGCTT TCCACTGTGG AG -             #AGGCTATG   3720                                                                  - - CCCCCTCACA ACGTTTTGGG GGCAGGAACT GGGAGAGATG CGGCCTGTGC CA -             #TTCTCTCC   3780                                                                  - - TCACACCTCC ATGCACACAG ACAATGCCCT GTACGGGAAA CAGGACTGTT GA -             #TAAGGGGG   3840                                                                  - - AGGCAAGAGG ACACCAGGCA AGGAGCAGTG GCTCTGAGCA AAAGAAAATA TT -             #TATTAAAT   3900                                                                  - - AAAACAAAAG TTTTCTCTGC CCTT          - #                  - #                   3924                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1098 amino - #acids                                                (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                               - - Arg Gly Lys Gly Arg Ala Ala Glu Glu Leu Gl - #u Pro Gly Thr Gly Thr       1               5   - #                10  - #                15                - - Val Glu Asp Gly Asp Gly Ser Glu Asp Gly Gl - #y Ser Ser Val Ala Ser                   20      - #            25      - #            30                    - - Gly Ser Gly Thr Gly Thr Val Leu Ser Leu Gl - #y Ala Cys Cys Leu Ala               35          - #        40          - #        45                        - - Leu Leu Gln Ile Phe Arg Ser Lys Lys Phe Pr - #o Ser Asp Lys Leu Glu           50              - #    55              - #    60                            - - Arg Leu Tyr Gln Arg Tyr Phe Phe Arg Leu As - #n Gln Ser Ser Leu Thr       65                  - #70                  - #75                  - #80         - - Met Leu Met Ala Val Leu Val Leu Val Cys Le - #u Val Met Leu Ala Phe                       85  - #                90  - #                95                - - His Ala Ala Arg Pro Pro Leu Gln Val Val Ty - #r Leu Ala Val Leu Ala                   100      - #           105      - #           110                   - - Ala Ala Val Gly Val Ile Leu Ile Met Ala Va - #l Leu Cys Asn Arg Ala               115          - #       120          - #       125                       - - Ala Phe His Gln Asp His Met Gly Leu Ala Cy - #s Tyr Ala Leu Ile Ala           130              - #   135              - #   140                           - - Val Val Leu Ala Val Gln Val Val Gly Leu Le - #u Leu Pro Gln Pro Arg       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Ser Ala Ser Glu Gly Ile Trp Trp Thr Val Ph - #e Phe Ile Tyr Thr         Ile                                                                                              165  - #               170  - #               175              - - Tyr Thr Leu Leu Pro Val Arg Met Arg Ala Al - #a Val Leu Ser Gly Val                   180      - #           185      - #           190                   - - Leu Leu Ser Ala Leu His Leu Ala Ile Ser Le - #u His Thr Asn Ala Gln               195          - #       200          - #       205                       - - Asp Gln Phe Leu Leu Lys Gln Leu Val Ser As - #n Val Leu Ile Phe Ser           210              - #   215              - #   220                           - - Cys Thr Asn Ile Val Gly Val Cys Thr His Ty - #r Pro Ala Glu Val Ser       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Gln Arg Gln Ala Phe Gln Glu Thr Arg Glu Cy - #s Ile Gln Ala Arg         Leu                                                                                              245  - #               250  - #               255              - - His Ser Gln Arg Glu Asn Gln Gln Gln Glu Ar - #g Leu Leu Leu Ser Val                   260      - #           265      - #           270                   - - Leu Pro Arg His Val Ala Met Glu Met Lys Al - #a Asp Ile Asn Ala Lys               275          - #       280          - #       285                       - - Gln Glu Asp Met Met Phe His Lys Ile Tyr Il - #e Gln Lys His Asp Asn           290              - #   295              - #   300                           - - Val Ser Ile Leu Phe Ala Asp Ile Glu Gly Ph - #e Thr Ser Leu Ala Ser       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Gln Cys Thr Ala Gln Glu Leu Val Met Thr Le - #u Asn Glu Leu Phe         Ala                                                                                              325  - #               330  - #               335              - - Arg Phe Asp Lys Leu Ala Ala Glu Asn His Cy - #s Leu Arg Ile Lys Ile                   340      - #           345      - #           350                   - - Leu Gly Asp Cys Tyr Tyr Cys Val Ser Gly Le - #u Pro Glu Ala Arg Ala               355          - #       360          - #       365                       - - Asp His Ala His Cys Cys Val Glu Met Gly Me - #t Asp Met Ile Glu Ala           370              - #   375              - #   380                           - - Ile Ser Ser Val Arg Glu Val Thr Gly Val As - #n Val Asn Met Arg Val       385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - Gly Ile His Ser Gly Arg Val His Cys Gly Va - #l Leu Gly Leu Arg         Lys                                                                                              405  - #               410  - #               415              - - Trp Gln Phe Asp Val Trp Ser Asn Asp Val Th - #r Leu Ala Asn His Met                   420      - #           425      - #           430                   - - Glu Ala Gly Gly Lys Ala Gly Arg Ile His Il - #e Thr Lys Ala Thr Leu               435          - #       440          - #       445                       - - Asn Tyr Leu Asn Gly Asp Tyr Glu Val Glu Pr - #o Gly Cys Gly Gly Glu           450              - #   455              - #   460                           - - Arg Asn Ala Tyr Leu Lys Glu His Ser Ile Gl - #u Thr Phe Leu Ile Leu       465                 4 - #70                 4 - #75                 4 -       #80                                                                               - - Arg Cys Thr Gln Lys Arg Lys Glu Glu Lys Al - #a Met Ile Ala Lys         Met                                                                                              485  - #               490  - #               495              - - Asn Arg Gln Arg Thr Asn Ser Ile Gly His As - #n Pro Pro His Trp Gly                   500      - #           505      - #           510                   - - Ala Glu Arg Pro Phe Tyr Asn His Leu Gly Gl - #y Asn Gln Val Ser Lys               515          - #       520          - #       525                       - - Glu Met Lys Arg Met Gly Phe Glu Asp Pro Ly - #s Asp Lys Asn Ala Gln           530              - #   535              - #   540                           - - Glu Ser Ala Asn Pro Glu Asp Glu Val Asp Gl - #u Phe Leu Gly Arg Ala       545                 5 - #50                 5 - #55                 5 -       #60                                                                               - - Ile Asp Ala Arg Ser Ile Asp Arg Leu Arg Se - #r Glu His Val Arg         Lys                                                                                              565  - #               570  - #               575              - - Phe Leu Leu Thr Phe Arg Glu Pro Asp Leu Gl - #u Lys Lys Tyr Ser Lys                   580      - #           585      - #           590                   - - Gln Val Asp Asp Arg Phe Gly Ala Tyr Val Al - #a Cys Ala Ser Leu Val               595          - #       600          - #       605                       - - Phe Leu Phe Ile Cys Phe Val Gln Ile Thr Il - #e Val Pro His Ser Leu           610              - #   615              - #   620                           - - Phe Met Leu Ser Phe Tyr Leu Ser Cys Phe Le - #u Leu Leu Ala Leu Val       625                 6 - #30                 6 - #35                 6 -       #40                                                                               - - Val Phe Ile Ser Val Ile Tyr Ala Cys Val Ly - #s Leu Phe Pro Thr         Pro                                                                                              645  - #               650  - #               655              - - Leu Gln Thr Leu Ser Arg Lys Ile Val Arg Se - #r Lys Lys Asn Ser Thr                   660      - #           665      - #           670                   - - Leu Val Gly Val Phe Thr Ile Thr Leu Val Ph - #e Leu Ser Ala Phe Val               675          - #       680          - #       685                       - - Asn Met Phe Met Cys Asn Ser Lys Asn Leu Va - #l Gly Cys Leu Ala Glu           690              - #   695              - #   700                           - - Glu His Asn Ile Thr Val Asn Gln Val Asn Al - #a Cys His Val Met Glu       705                 7 - #10                 7 - #15                 7 -       #20                                                                               - - Ser Ala Phe Asn Tyr Ser Leu Gly Asp Glu Gl - #n Gly Phe Cys Gly         Ser                                                                                              725  - #               730  - #               735              - - Pro Gln Ser Asn Cys Asn Phe Pro Glu Tyr Ph - #e Thr Tyr Ser Val Leu                   740      - #           745      - #           750                   - - Leu Ser Leu Leu Ala Cys Ser Val Phe Leu Gl - #n Ile Ser Cys Ile Gly               755          - #       760          - #       765                       - - Lys Leu Val Leu Met Leu Ala Ile Glu Leu Il - #e Tyr Val Leu Ile Val           770              - #   775              - #   780                           - - Glu Val Pro Gly Val Thr Leu Phe Asp Asn Al - #a Asp Leu Leu Val Thr       785                 7 - #90                 7 - #95                 8 -       #00                                                                               - - Ala Asn Ala Ile Asp Phe Ser Asn Asn Gly Th - #r Ser Gln Cys Pro         Glu                                                                                              805  - #               810  - #               815              - - His Ala Thr Lys Val Ala Leu Lys Val Val Th - #r Pro Ile Ile Ile Ser                   820      - #           825      - #           830                   - - Val Phe Val Leu Ala Leu Tyr Leu His Ala Gl - #n Gln Val Glu Ser Thr               835          - #       840          - #       845                       - - Ala Arg Leu Asp Phe Leu Trp Lys Leu Gln Al - #a Thr Glu Glu Lys Glu           850              - #   855              - #   860                           - - Glu Met Glu Glu Leu Gln Ala Tyr Asn Arg Ar - #g Leu Leu His Asn Ile       865                 8 - #70                 8 - #75                 8 -       #80                                                                               - - Leu Pro Lys Asp Val Ala Ala His Phe Leu Al - #a Arg Glu Arg Arg         Asn                                                                                              885  - #               890  - #               895              - - Asp Glu Leu Tyr Tyr Gln Ser Cys Glu Cys Va - #l Ala Val Met Phe Ala                   900      - #           905      - #           910                   - - Ser Ile Ala Asn Phe Ser Glu Phe Tyr Val Gl - #u Leu Glu Ala Asn Asn               915          - #       920          - #       925                       - - Glu Gly Val Glu Cys Leu Arg Leu Leu Asn Gl - #u Ile Ile Ala Asp Phe           930              - #   935              - #   940                           - - Asp Glu Ile Ile Ser Glu Asp Arg Phe Arg Gl - #n Leu Glu Lys Ile Lys       945                 9 - #50                 9 - #55                 9 -       #60                                                                               - - Thr Ile Gly Ser Thr Tyr Met Ala Ala Ser Gl - #y Leu Asn Asp Ser         Thr                                                                                              965  - #               970  - #               975              - - Tyr Asp Lys Ala Gly Lys Thr His Ile Lys Al - #a Leu Ala Asp Phe Ala                   980      - #           985      - #           990                   - - Met Lys Leu Met Asp Gln Met Lys Tyr Ile As - #n Glu His Ser Phe Asn               995          - #       1000          - #      1005                      - - Asn Phe Gln Met Lys Ile Gly Leu Asn Ile Gl - #y Pro Val Val Ala Gly           1010             - #   1015              - #  1020                          - - Val Ile Gly Ala Arg Lys Pro Gln Tyr Asp Il - #e Trp Gly Asn Thr Val       1025                1030 - #                1035 - #               1040         - - Asn Val Ala Ser Arg Met Asp Ser Thr Gly Va - #l Pro Asp Arg Ile Gln                       1045 - #               1050  - #              1055              - - Val Thr Thr Asp Met Tyr Gln Val Leu Ala Al - #a Asn Thr Tyr Gln Leu                   1060     - #           1065      - #          1070                  - - Glu Cys Arg Gly Val Val Lys Val Lys Gly Ly - #s Gly Glu Met Met Thr               1075         - #       1080          - #      1085                      - - Tyr Phe Leu Asn Gly Gly Pro Pro Leu Ser                                       1090             - #   1095                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 4131 base - #pairs                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                               - - GGCGGCACCA AGAATGCCCC TGCCCGTGGC CCGATCCGGT TCTGGGCGCA GC -              #AGCATGTC     60                                                                  - - ATGGTTTAGC GGCCTCCTGG TTCCCAAAGT GGATGAACGG AAAACAGCCT GG -             #GGCGAACG    120                                                                  - - CAATGGACAG AAGCGCCCAC GCCAGGCGAC CCGAGCCCGT GGCTTCTGCG CG -             #CCCCGCTA    180                                                                  - - CATGAGCTGC CTCAAGAATG TGGAGCCACC CAGCCCCACT CCTGCAGCTC GC -             #ACTCGGTG    240                                                                  - - CCCCTGGCAG GATGAAGCCT TCATCAGGAG GGCTGGCCCG GGAAGGGGTG TG -             #GAGCTGGG    300                                                                  - - GCTGCGGTCA GTGGCCTTGG GTTTTGATGA CACTGAGGTG ACCACACCGA TG -             #GGGACAGC    360                                                                  - - TGAAGTGGCA CCCGACACAT CGCCTCGAAG CGGTCCGTCC TGCTGGCACC GG -             #CTAGCGCA    420                                                                  - - GGTGTTCCAG TCTAAGCAGT TCCCGTCCGC CAAGCTGGAG CGTCTGTACC AG -             #CGGTACTT    480                                                                  - - CTTCCAGATG AACCAGAGCA GCCTCACGCT GCTCATGGCG GTGCTTGTGC TC -             #CTCATGGC    540                                                                  - - TGTACTGTTG ACCTTCCACG CCGCGCCTGC CCTGCCTCAG CCTGCTTATG TG -             #GCCCTGCT    600                                                                  - - GACCTGTGCC TCCGTCCTTT TTGTGGTACT CATGGTAGTG TGTAACCGAC AT -             #AGCTTCCG    660                                                                  - - CCAGGACTCC ATGTGGGTAG TGAGCTATGT GGTTCTGGGC ATCCTAGCAG CC -             #GTGCAAGT    720                                                                  - - CGGGGGTGCC CTGGCAGCCA ACCCACGCAG CCCCTCAGCA GGCCTTTGGT GC -             #CCCGTGTT    780                                                                  - - CTTCGTCTAC ATCACCTACA CACTTCTTCC CATTCGTATG CGAGCGGCCG TG -             #CTCAGTGG    840                                                                  - - CCTGGGTCTT TCCACCCTGC ATTTGATTTT GGCCTGGCAT CTCAACAATG GT -             #GACCCCTT    900                                                                  - - CCTTTGGAAG CAGCTCGGTG CTAACGTGGT GCTCTTCCTG TGCACCAATG CC -             #ATCGGTGT    960                                                                  - - CTGCACGCAC TACCCCGCTG AAGTGTCTCA GCGCCAAGCC TTTCAGGAGA CC -             #CGTGGTTA   1020                                                                  - - CATCCAGGCC CGGCTGCACT TGCAGCATGA GAATCGACAG CAGGAACGGC TG -             #CTGCTGTC   1080                                                                  - - GGTGTTGCCC CAGCATGTTG CCATGGAAAT GAAAGAGGAT ATCAACACAA AA -             #AAGGAAGA   1140                                                                  - - CATGATGTTC CACAAGATTT ACATCCAGAA GCATGACAAT GTCAGCATCC TG -             #TTTGCCGA   1200                                                                  - - CATCGAGGGC TTCACCAGCC TGGCCTCCCA GTGCACTGCC CAGGAACTGG TC -             #ATGACCTT   1260                                                                  - - GAATGAGCTC TTTGCCCGGT TCGACAAGCT GGCTGCGGAG AATCACTGTC TG -             #AGGATCAA   1320                                                                  - - GATCTTAGGA GACTGTTACT ACTGTGTGTC GGGGCTGCCG GAGGCCCGGG CA -             #GACCATGC   1380                                                                  - - CCACTGCTGT GTGGAGATGG GGGTAGACAT GATCGAGGCC ATCTCGCTGG TG -             #CGTGAGGT   1440                                                                  - - AACGGGTGTA AATGTGAACA TGCGCGTGGG CATCCACAGC GGGCGTGTAC AC -             #TGCGGTGT   1500                                                                  - - CCTTGGTCTG CGGAAATGGC AGTTTGATGT CTGGTCCAAC GATGTGACCC TG -             #GCCAACCA   1560                                                                  - - CATGGAGGCG GGGGGCCGGG CGGGCCGCAT CCACATCACT CGGGCCACAC TG -             #CAGTACCT   1620                                                                  - - GAACGGGGAC TATGAGGTGG AGCCAGGCCG TGGCGGTGAG CGCAACGCGT AC -             #CTCAAGGA   1680                                                                  - - GCAGTGCATT GAGACCTTCC TCATACTAGG AGCCAGCCAG AAACGGAAAG AG -             #GAGAAGGC   1740                                                                  - - CATGCTGGTC AAGCTGCAGC GGACGCGGGC CAACTCCATG GAAGGACTGA TG -             #CCCCGCTG   1800                                                                  - - GGTTCCTGAC CGTGCCTTCT CCCGGACCAA GGACTCTAAG GCATTCCGAC AG -             #ATGGGCAT   1860                                                                  - - CGATGACTCT AGCAAAGAGA ACCGGGGTGC CCAAGATGCT CTGAACCCTG AG -             #GATGAGGT   1920                                                                  - - GGACGAGTTT CTGGGCCGAG CCATCGATGC CCGAAGCATC GACCAGCTGC GT -             #AAGGACCA   1980                                                                  - - TGTGCGCCGG TTCCTGCTCA CCTTCCAGAG GGAGGATCTC GAGAAGAAGT AT -             #TCACGGAA   2040                                                                  - - AGTAGACCCT CGTTTCGGAG CCTACGTCGC CTGTGCCCTC CTGGTTTTCT GC -             #TTCATCTG   2100                                                                  - - TTTCATCCAG TTCCTCGTAT TCCCACACTC CGCCCTGATA CTCGGGATTT AT -             #GCCGGGAT   2160                                                                  - - CTTCCTTTTG CTGCTGGTCA CCGTGCTCAT CTGTGCTGTG TGCTCCTGTG GG -             #TCTTTCTT   2220                                                                  - - CCCCAACGCC CTGCAGCGCC TGTCCCGCAG TATCGTCCGC TCACGGGTGC AC -             #AGCACGGC   2280                                                                  - - TGTTGGAGTC TTCTCGGTTC TGCTTGTGTT CATCTCTGCC ATTGCCAACA TG -             #TTCACCTG   2340                                                                  - - CAGTCACACC CCACTGAGGA CCTGTGCGGC CCGGATGCTG AACTTAACAC CG -             #TCCGATGT   2400                                                                  - - CACCGCCTGC CACCTACGAC AGCTCAATTA CTCTCTGGGA CTGGAAGCTC CC -             #CTGTGTGA   2460                                                                  - - GGGCACCGCA CCCACCTGCA GCTTCCCTGA GTACTTTGTC GGGAGTGTGC TG -             #CTGAGTCT   2520                                                                  - - CTTGGCCAGC TCCGTCTTCC TCCACATCAG CAGCATTGGC AAGCTAGTTA TG -             #ACCTTTGT   2580                                                                  - - CTTGGGGTTC ATCTACTTGC TTCTGCTTTT GCTGGGTCCC CCAGCCACCA TC -             #TTTGACAA   2640                                                                  - - CTATGATCTA CTGCTTAGCG TCCATGGCTT GGCTTCCTCC AATGAGACCT TC -             #GATGGGCT   2700                                                                  - - GGACTGCCCA GCCGTAGGGA GGGTAGCGCT CAAATACATG ACCCCTGTGA TT -             #CTCCTCGT   2760                                                                  - - GTTCGCCCTG GCACTGTATC TACACGCACA ACAGGTGGAA TCTACCGCCC GC -             #CTGGACTT   2820                                                                  - - CCTGTGGAAA CTGCAGGCCA CAGGGGAGAA GGAGGAGATG GAGGAGTTGC AG -             #GCCTACAA   2880                                                                  - - CCGGCGGCTG CTGCATAACA TCCTTCCCAA GGACGTGGCT GCCCACTTCC TG -             #GCCCGGGA   2940                                                                  - - GCGCCGCAAC GACGAGCTGT ACTACCAATC CTGCGAGTGC GTGGCTGTCA TG -             #TTTGCCTC   3000                                                                  - - CATCGCCAAC TTCTCTGAGT TCTATGTGGA ACTGGAGGCG AACAATGAGG GC -             #GTGGAGTG   3060                                                                  - - CCTGCGACTG CTCAATGAGA TCATCGCGGA CTTTGATGAG ATCATCAGTG AG -             #GAGAGGTT   3120                                                                  - - CCGGCAGCTG GAGAAGATCA AGACCATCGG TAGCACTTAC ATGGCCGCCT CC -             #GGGCTAAA   3180                                                                  - - TGCCAGCACC TATGACCAGG TCGGCCGCTC GCACATCACC GCCCTGGCAG AC -             #TACGCCAT   3240                                                                  - - GCGGCTTATG GAGCAAATGA AACACATCAA CGAACACTCT TTCAACAACT TC -             #CAGATGAA   3300                                                                  - - GATCGGGTTG AACATGGGTC CGGTTGTAGC AGGTGTCATT GGGGCCCGGA AG -             #CCACAGTA   3360                                                                  - - TGACATCTGG GGAAACACGG TGAATGTTTC CAGCCGTATG GACAGCACAG GA -             #GTTCCTGA   3420                                                                  - - CCGAATACAG GTGACCACGG ATCTCTACCA GGTTCTAGCT GCCAAGGGCT AC -             #CAACTGGA   3480                                                                  - - GTGTCGAGGG GTGGTCAAGG TGAAGGGAAA GGGGGAGATG ACCACCTACT TC -             #CTCAATGG   3540                                                                  - - GGGCCCCAGC AGTTAGCAGA GCGCACGAGT GGAAATTCAA CCAAAGGGAC CA -             #AGGTGGGC   3600                                                                  - - ACTGAGTGGA CTTTTCTGCT CACTGGATGG AGCTGTGGCA GGGGGCTCTG AG -             #CCTTCAGG   3660                                                                  - - TCTTGCTGAC AGCAAAAGGG AACACCCCAG CAGGCTGTGC TTGGACCATA TC -             #CGTCTGCC   3720                                                                  - - TTCAGAGCAG CAAAGGAAGG GACGCCGAGA GGATTATTAT CCAAGTGACT TT -             #CTTAATCA   3780                                                                  - - GAGTAAGGCT GTTTGCTTTT TTTTCCCCCC CTTTGGACGC AGCTGAGAGC AG -             #AGCCTCCT   3840                                                                  - - GTTTGAGAGT AAAATGGCAA CTTACTCTGC CTACTGTTTC CCTGTCTGGG CG -             #ACAGGCTC   3900                                                                  - - AGGGCTGGGA CCTTTCTTTC CCTATTTTTC CTGGGACTAT TTTTGTACAA GA -             #GTGGGGCA   3960                                                                  - - GGCATGAGGA ATGCTTGCCT TCACTGGCCT GTGTCAGCAG CATTTGTCTT GG -             #GCACTCCC   4020                                                                  - - AGTACCAGCC AAGTCTCCTT CCTAGCACAA CAAGGCAGAG GAAGGAGGTG CT -             #GTGGGACC   4080                                                                  - - TAGCTCTGAC CAGATTTCAG GGGAATGTTT CCATTTGCCA AATCCTAGTC C - #                4131                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1180 amino - #acids                                                (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                               - - Met Pro Leu Pro Val Ala Arg Ser Gly Ser Gl - #y Arg Ser Ser Met Ser       1               5   - #                10  - #                15                - - Trp Phe Ser Gly Leu Leu Val Pro Lys Val As - #p Glu Arg Lys Thr Ala                   20      - #            25      - #            30                    - - Trp Gly Glu Arg Asn Gly Gln Lys Arg Pro Ar - #g Gln Ala Thr Arg Ala               35          - #        40          - #        45                        - - Arg Gly Phe Cys Ala Pro Arg Tyr Met Ser Cy - #s Leu Lys Asn Val Glu           50              - #    55              - #    60                            - - Pro Pro Ser Pro Thr Pro Ala Ala Arg Thr Ar - #g Cys Pro Trp Gln Asp       65                  - #70                  - #75                  - #80         - - Glu Ala Phe Ile Arg Arg Ala Gly Pro Gly Ar - #g Gly Val Glu Leu Gly                       85  - #                90  - #                95                - - Leu Arg Ser Val Ala Leu Gly Phe Asp Asp Th - #r Glu Val Thr Thr Pro                   100      - #           105      - #           110                   - - Met Gly Thr Ala Glu Val Ala Pro Asp Thr Se - #r Pro Arg Ser Gly Pro               115          - #       120          - #       125                       - - Ser Cys Trp His Arg Leu Ala Gln Val Phe Gl - #n Ser Lys Gln Phe Pro           130              - #   135              - #   140                           - - Ser Ala Lys Leu Glu Arg Leu Tyr Gln Arg Ty - #r Phe Phe Gln Met Asn       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Gln Ser Ser Leu Thr Leu Leu Met Ala Val Le - #u Val Leu Leu Met         Ala                                                                                              165  - #               170  - #               175              - - Val Leu Leu Thr Phe His Ala Ala Pro Ala Le - #u Pro Gln Pro Ala Tyr                   180      - #           185      - #           190                   - - Val Ala Leu Leu Thr Cys Ala Ser Val Leu Ph - #e Val Val Leu Met Val               195          - #       200          - #       205                       - - Val Cys Asn Arg His Ser Phe Arg Gln Asp Se - #r Met Trp Val Val Ser           210              - #   215              - #   220                           - - Tyr Val Val Leu Gly Ile Leu Ala Ala Val Gl - #n Val Gly Gly Ala Leu       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Ala Ala Asn Pro Arg Ser Pro Ser Ala Gly Le - #u Trp Cys Pro Val         Phe                                                                                              245  - #               250  - #               255              - - Phe Val Tyr Ile Thr Tyr Thr Leu Leu Pro Il - #e Arg Met Arg Ala Ala                   260      - #           265      - #           270                   - - Val Leu Ser Gly Leu Gly Leu Ser Thr Leu Hi - #s Leu Ile Leu Ala Trp               275          - #       280          - #       285                       - - His Leu Asn Asn Gly Asp Pro Phe Leu Trp Ly - #s Gln Leu Gly Ala Asn           290              - #   295              - #   300                           - - Val Val Leu Phe Leu Cys Thr Asn Ala Ile Gl - #y Val Cys Thr His Tyr       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Pro Ala Glu Val Ser Gln Arg Gln Ala Phe Gl - #n Glu Thr Arg Gly         Tyr                                                                                              325  - #               330  - #               335              - - Ile Gln Ala Arg Leu His Leu Gln His Glu As - #n Arg Gln Gln Glu Arg                   340      - #           345      - #           350                   - - Leu Leu Leu Ser Val Leu Pro Gln His Val Al - #a Met Glu Met Lys Glu               355          - #       360          - #       365                       - - Asp Ile Asn Thr Lys Lys Glu Asp Met Met Ph - #e His Lys Ile Tyr Ile           370              - #   375              - #   380                           - - Gln Lys His Asp Asn Val Ser Ile Leu Phe Al - #a Asp Ile Glu Gly Phe       385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - Thr Ser Leu Ala Ser Gln Cys Thr Ala Gln Gl - #u Leu Val Met Thr         Leu                                                                                              405  - #               410  - #               415              - - Asn Glu Leu Phe Ala Arg Phe Asp Lys Leu Al - #a Ala Glu Asn His Cys                   420      - #           425      - #           430                   - - Leu Arg Ile Lys Ile Leu Gly Asp Cys Tyr Ty - #r Cys Val Ser Gly Leu               435          - #       440          - #       445                       - - Pro Glu Ala Arg Ala Asp His Ala His Cys Cy - #s Val Glu Met Gly Val           450              - #   455              - #   460                           - - Asp Met Ile Glu Ala Ile Ser Leu Val Arg Gl - #u Val Thr Gly Val Asn       465                 4 - #70                 4 - #75                 4 -       #80                                                                               - - Val Asn Met Arg Val Gly Ile His Ser Gly Ar - #g Val His Cys Gly         Val                                                                                              485  - #               490  - #               495              - - Leu Gly Leu Arg Lys Trp Gln Phe Asp Val Tr - #p Ser Asn Asp Val Thr                   500      - #           505      - #           510                   - - Leu Ala Asn His Met Glu Ala Gly Gly Arg Al - #a Gly Arg Ile His Ile               515          - #       520          - #       525                       - - Thr Arg Ala Thr Leu Gln Tyr Leu Asn Gly As - #p Tyr Glu Val Glu Pro           530              - #   535              - #   540                           - - Gly Arg Gly Gly Glu Arg Asn Ala Tyr Leu Ly - #s Glu Gln Cys Ile Glu       545                 5 - #50                 5 - #55                 5 -       #60                                                                               - - Thr Phe Leu Ile Leu Gly Ala Ser Gln Lys Ar - #g Lys Glu Glu Lys         Ala                                                                                              565  - #               570  - #               575              - - Met Leu Val Lys Leu Gln Arg Thr Arg Ala As - #n Ser Met Glu Gly Leu                   580      - #           585      - #           590                   - - Met Pro Arg Trp Val Pro Asp Arg Ala Phe Se - #r Arg Thr Lys Asp Ser               595          - #       600          - #       605                       - - Lys Ala Phe Arg Gln Met Gly Ile Asp Asp Se - #r Ser Lys Glu Asn Arg           610              - #   615              - #   620                           - - Gly Ala Gln Asp Ala Leu Asn Pro Glu Asp Gl - #u Val Asp Glu Phe Leu       625                 6 - #30                 6 - #35                 6 -       #40                                                                               - - Gly Arg Ala Ile Asp Ala Arg Ser Ile Asp Gl - #n Leu Arg Lys Asp         His                                                                                              645  - #               650  - #               655              - - Val Arg Arg Phe Leu Leu Thr Phe Gln Arg Gl - #u Asp Leu Glu Lys Lys                   660      - #           665      - #           670                   - - Tyr Ser Arg Lys Val Asp Pro Arg Phe Gly Al - #a Tyr Val Ala Cys Ala               675          - #       680          - #       685                       - - Leu Leu Val Phe Cys Phe Ile Cys Phe Ile Gl - #n Phe Leu Val Phe Pro           690              - #   695              - #   700                           - - His Ser Ala Leu Ile Leu Gly Ile Tyr Ala Gl - #y Ile Phe Leu Leu Leu       705                 7 - #10                 7 - #15                 7 -       #20                                                                               - - Leu Val Thr Val Leu Ile Cys Ala Val Cys Se - #r Cys Gly Ser Phe         Phe                                                                                              725  - #               730  - #               735              - - Pro Asn Ala Leu Gln Arg Leu Ser Arg Ser Il - #e Val Arg Ser Arg Val                   740      - #           745      - #           750                   - - His Ser Thr Ala Val Gly Val Phe Ser Val Le - #u Leu Val Phe Ile Ser               755          - #       760          - #       765                       - - Ala Ile Ala Asn Met Phe Thr Cys Ser His Th - #r Pro Leu Arg Thr Cys           770              - #   775              - #   780                           - - Ala Ala Arg Met Leu Asn Leu Thr Pro Ser As - #p Val Thr Ala Cys His       785                 7 - #90                 7 - #95                 8 -       #00                                                                               - - Leu Arg Gln Leu Asn Tyr Ser Leu Gly Leu Gl - #u Ala Pro Leu Cys         Glu                                                                                              805  - #               810  - #               815              - - Gly Thr Ala Pro Thr Cys Ser Phe Pro Glu Ty - #r Phe Val Gly Ser Val                   820      - #           825      - #           830                   - - Leu Leu Ser Leu Leu Ala Ser Ser Val Phe Le - #u His Ile Ser Ser Ile               835          - #       840          - #       845                       - - Gly Lys Leu Val Met Thr Phe Val Leu Gly Ph - #e Ile Tyr Leu Leu Leu           850              - #   855              - #   860                           - - Leu Leu Leu Gly Pro Pro Ala Thr Ile Phe As - #p Asn Tyr Asp Leu Leu       865                 8 - #70                 8 - #75                 8 -       #80                                                                               - - Leu Ser Val His Gly Leu Ala Ser Ser Asn Gl - #u Thr Phe Asp Gly         Leu                                                                                              885  - #               890  - #               895              - - Asp Cys Pro Ala Val Gly Arg Val Ala Leu Ly - #s Tyr Met Thr Pro Val                   900      - #           905      - #           910                   - - Ile Leu Leu Val Phe Ala Leu Ala Leu Tyr Le - #u His Ala Gln Gln Val               915          - #       920          - #       925                       - - Glu Ser Thr Ala Arg Leu Asp Phe Leu Trp Ly - #s Leu Gln Ala Thr Gly           930              - #   935              - #   940                           - - Glu Lys Glu Glu Met Glu Glu Leu Gln Ala Ty - #r Asn Arg Arg Leu Leu       945                 9 - #50                 9 - #55                 9 -       #60                                                                               - - His Asn Ile Leu Pro Lys Asp Val Ala Ala Hi - #s Phe Leu Ala Arg         Glu                                                                                              965  - #               970  - #               975              - - Arg Arg Asn Asp Glu Leu Tyr Tyr Gln Ser Cy - #s Glu Cys Val Ala Val                   980      - #           985      - #           990                   - - Met Phe Ala Ser Ile Ala Asn Phe Ser Glu Ph - #e Tyr Val Glu Leu Glu               995          - #       1000          - #      1005                      - - Ala Asn Asn Glu Gly Val Glu Cys Leu Arg Le - #u Leu Asn Glu Ile Ile           1010             - #   1015              - #  1020                          - - Ala Asp Phe Asp Glu Ile Ile Ser Glu Glu Ar - #g Phe Arg Gln Leu Glu       1025                1030 - #                1035 - #               1040         - - Lys Ile Lys Thr Ile Gly Ser Thr Tyr Met Al - #a Ala Ser Gly Leu Asn                       1045 - #               1050  - #              1055              - - Ala Ser Thr Tyr Asp Gln Val Gly Arg Ser Hi - #s Ile Thr Ala Leu Ala                   1060     - #           1065      - #          1070                  - - Asp Tyr Ala Met Arg Leu Met Glu Gln Met Ly - #s His Ile Asn Glu His               1075         - #       1080          - #      1085                      - - Ser Phe Asn Asn Phe Gln Met Lys Ile Gly Le - #u Asn Met Gly Pro Val           1090             - #   1095              - #  1100                          - - Val Ala Gly Val Ile Gly Ala Arg Lys Pro Gl - #n Tyr Asp Ile Trp Gly       1105                1110 - #                1115 - #               1120         - - Asn Thr Val Asn Val Ser Ser Arg Met Asp Se - #r Thr Gly Val Pro Asp                       1125 - #               1130  - #              1135              - - Arg Ile Gln Val Thr Thr Asp Leu Tyr Gln Va - #l Leu Ala Ala Lys Gly                   1140     - #           1145      - #          1150                  - - Tyr Gln Leu Glu Cys Arg Gly Val Val Lys Va - #l Lys Gly Lys Gly Glu               1155         - #       1160          - #      1165                      - - Met Thr Thr Tyr Phe Leu Asn Gly Gly Pro Se - #r Ser                           1170             - #   1175              - #  1180                          - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 5199 base - #pairs                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                               - - GTGGCTCCCT CCTGCAGCCT CAGGGACACC GGAGATGGCC CAGTGCGGCA GG -              #TAACAGGG     60                                                                  - - TCGGAGGCGG CTACGCGGGG GACCGTGTGG GGACGTTCCC GAGATCCCCA GC -             #CCGCGCCC    120                                                                  - - CGCACGGTCA GTGTGACCAG CTGGGGGCGC CCCGGCTCTC GCGCTCGCGG GC -             #GGGAACTC    180                                                                  - - CAGAGCGCCC GGGCCCTCGG CGGTGGCGGC AGGGAAGAAG ATGTCTCTAC CT -             #GGAAGCTC    240                                                                  - - CCAGGAGACA GCTGTGGAGC CCACGAGAGC CACAGGGCCT TGACCTCATC TC -             #TTCCCACC    300                                                                  - - ATCCAAGAGG GCAAAGTCAG GGGCTTTAAA CGGGAGAGGA GAGACGACTC AG -             #CACTCCTT    360                                                                  - - GTGATAAGGC CCTGTGGATG TGTGGCTAGC TTGCTCTGTC CTGTGCGTCA AG -             #GAATGCAG    420                                                                  - - GTGCAAAGAG CTGAGGACCT GGAAGTTTCT GTCAGCTCCT CCTAGACACC CT -             #GAAGACAC    480                                                                  - - CAGTGAACAG AACCCAGTGG CTGGAGCAGG TGTCACATGC TCATGGAGCC AC -             #GGGCTCTA    540                                                                  - - GGTGCCTCTG TCCAGTCCTG CCTGATACCC TCAGAAAGGG AAGAAGCCTA GG -             #CCACACAC    600                                                                  - - TAAAGCGTCA GCTGACAGAT GTATCTCAGA CCGTGGGGCT CCTGAGCAGG CT -             #GCTGGACC    660                                                                  - - AGGCCACTGG GGAAGAGGAT GCCAGCCAAG GGGCGCTACT TCCTAAATGA GG -             #GTGATGAA    720                                                                  - - GGCCCCGACC AGGCAGCGCT CTATGAGAAG TACCGGCTCA CCAGCTTGCA CG -             #GGCCACTG    780                                                                  - - CTGCTCTTGC TCCTCCTGGT GGCCGCGGCC ACCTGCATTG CGCTCATCAG CA -             #TCGCCTTC    840                                                                  - - AGTCATGAGG ATCTCCGCAG ACACCAGGTT GTCCTGGGCA CTGCGTTCCT CA -             #TGCTGACG    900                                                                  - - CTGTTTGTGG CTCTCTATGT GCTGGTGTAT GTCGAGTGCC TGGTGCAGCG GT -             #GGCTGCGG    960                                                                  - - GCCTTGGCGC TACTCACCTG GGCTTGCCTC ATGGTACTAG GCTCCGTGCT GA -             #TGTGGGAC   1020                                                                  - - TCTTTGGAGA ATGAAGCCCA TGCGTGGGAG CAGGTGCCTT TCTTCCTGTT TG -             #TCGTCTTT   1080                                                                  - - GTGGTGTATG CACTACTGCC TCTCAGCAGG AGGGCAGCCA TCGTGGTAGG CG -             #TGACCTCC   1140                                                                  - - ACGGTCTCCC ATCTCCTGGT GTTTGGAGCT GTGACAAGAG CCTTCCAGAC GT -             #CCATGTCT   1200                                                                  - - AGCACTCAAC TGGGGCTGCA GCTCCTGGCC AATGCCGTTA TTCTCCTGGG TG -             #GGAACTTC   1260                                                                  - - ACGGGTGCCT TCCACAAGCA CCAGCTGCAG GACGCGTCCA GGGATCTCTT TA -             #TCTACACC   1320                                                                  - - GTCAAATGCA TCCAGATCCG TCGGAAGCTT CGTGTGGAGA AGCGCCAACA GG -             #AGAACCTG   1380                                                                  - - CTTCTGTCAG TGCTCCCAGC ACACATCTCC ATGGGTATGA AGCTGGCCAT CA -             #TTGAGCGC   1440                                                                  - - CTCAAAGAGG GTGGTGACCG ACACTACATG CCCGACAACA ACTTTCACAG CC -             #TCTATGTC   1500                                                                  - - AAGCGGCACC AGAATGTCAG CATCTTGTAT GCAGACATCG TGGGCTTCAC GA -             #GGCTGGCC   1560                                                                  - - AGCGACTGCT CTCCCAAGGA GCTGGTGGTG GTGCTCAACG AGCTGTTTGG GA -             #AGTTTGAC   1620                                                                  - - CAGATTGCTA AGGCCAATGA GTGCATGCGG ATCAAGATCC TGGGTGACTG TT -             #ACTACTGC   1680                                                                  - - GTGTCAGGCC TGCCCGTATC GCTGCCCACA CATGCCCGCA ACTGTGTGAA GA -             #TGGGTCTG   1740                                                                  - - GACATCTGCG AGGCCATTAA GCAGGTGCGT GAGGCCACGG GCGTGGACAT CA -             #GCATGCGT   1800                                                                  - - GTGGGCATTC ACTCCGGGAA TGTGCTATGT GGGGTCATCG GGCTCCGTAA GT -             #GGCAGTAT   1860                                                                  - - GATGTGTGGT CCCATGATGT GTCCCTGGCC AACAGGATGG AGGCAGCTGG AG -             #TCCCTGGC   1920                                                                  - - CGGGTGCACA TCACAGAGGC AACATTGAAT CACCTGGACA AGGCATACGA GG -             #TGGAGGAT   1980                                                                  - - GGGCATGGGG AGCAGCGAGA CCCCTATCTG AAAGAGATGA ACATCCGAAC CT -             #ACCTGGTG   2040                                                                  - - ATCGATCCCC GGAGCCAGCA GCCACCCCCA CCCAGCCACC ACCTCTCCAA GC -             #CCAAGGGG   2100                                                                  - - GACGCAACTC TGAAGATGCG GGCTTCAGTG CGTGTAACCC GCTATCTGGA GT -             #CTTGGGGG   2160                                                                  - - GCAGCACGGC CCTTTGCACA CCTCAACCAC CGGGAGAGTG TGAGCAGCAG TG -             #AGACCCCC   2220                                                                  - - ATCTCCAATG GACGGAGGCA GAAGGCCATT CCTCTGCGTC GACACCGTGC CC -             #CTGATAGG   2280                                                                  - - AGTGCATCCC CCAAGGGGCG CTTGGAAGAT GACTGTGATG ACGAGATGCT GT -             #CAGCCATT   2340                                                                  - - GAGGGTCTCA GCTCCACCAG GCCCTGCTGC TCCAAGTCTG ATGACTTCCA CA -             #CCTTTGGT   2400                                                                  - - CCCATCTTCT TGGAGAAGGG CTTTGAGCGT GAGTACCGCC TGGTGCCCAT CC -             #CCCGGGCT   2460                                                                  - - CGCTACGACT TCGCCTGTGC CAGCCTTGTC TTCGTCTGCA TCCTGCTTGT CC -             #ACCTTCTA   2520                                                                  - - GTGATGCCCA GGATGGCAAC TCTGGGTGTG TCCTTTGGGT TGGTGGCCTG CC -             #TGCTGGGG   2580                                                                  - - CTGGTTCTGA GTTTCTGCTT TGCTACTGAG TTCTCGAGGT GCTTTCCATC CC -             #GAAGTACA   2640                                                                  - - CTCCAGGCCA TCTCGGAGAG CGTGGAGACG CAGCCCCTGG TCAGGCTTGT CC -             #TGGTTGTG   2700                                                                  - - CTGACTGTTG GCAGCCTACT GACTGTCGCC ATCATTAACA TGCCACTGAC GC -             #TTAACCCA   2760                                                                  - - GGCCCAGAGC AGCCTGGAGA CAACAAGACA AGCCCACTGG CTGCACAGAA CA -             #GAGTTGGG   2820                                                                  - - ACCCCATATG AGCTCCTCCC GTACTACACC TGCAGCTGCA TCCTGGGCTT CA -             #TTGCATGC   2880                                                                  - - TCTGTTTTCC TGCGGATGAG CCTAGAGCTG AAGGCCATGC TGCTGACAGT GG -             #CCTTGGTG   2940                                                                  - - GCCTACCTGC TGCTCTTCAA CCTCTCCCCA TGCTGGCACG TCTCAGGCAA CA -             #GCACTGAG   3000                                                                  - - ACCAACGGGA CACAAAGGAC ACGGCTGCTC CTGTCTGATG CACAAAGCAT GC -             #CCAGCCAC   3060                                                                  - - ACCCTTGCTC CGGGGGCTCA GGAGACTGCC CCTTCTCCCA GTTATTTAGA GA -             #GAGACCTG   3120                                                                  - - AAGATCATGG TTAACTTCTA CCTGATCCTG TTCTATGCCA CCCTCATCTT GC -             #TGTCTAGA   3180                                                                  - - CAGATTGACT ACTACTGCCG CTTGGACTGT CTGTGGAAGA AGAAGTTCAA AA -             #AGGAGCAC   3240                                                                  - - GAGGAGTTTG AAACAATGGA GAATGTGAAC CGCCTCCTCC TGGAGAATGT GC -             #TGCCGGCG   3300                                                                  - - CACGTGGCTG CCCACTTCAT TGGGGACAAG GCAGCAGAGG ATTGGTACCA TC -             #AATCTTAT   3360                                                                  - - GACTGTGTCT GTGTCATGTT TGCATCCGTT CCGGACTTCA AAGTGTTCTA CA -             #CTGAGTGT   3420                                                                  - - GATGTCAACA AAGAAGGACT GGAGTGCCTT CGACTGCTGA ATGAGATAAT TG -             #CTGATTTT   3480                                                                  - - GACGAGCTCC TGCTGAAGCC CAAGTTCAGT GGTGTGGAGA AGATCAAGAC CA -             #TTGGCAGC   3540                                                                  - - ACCTACATGG CGGCAGCAGG GCTCAGTGCC CCCTCAGGAC ATGAGAACCA GG -             #ACCTGGAG   3600                                                                  - - CGGAAGCACG TGCACATCGG AGTCTTGGTA GAATTTAGCA TGGCCCTGAT GA -             #GCAAGCTG   3660                                                                  - - GATGGGATCA ACAGGCACTC CTTCAACTCC TTCCGCCTCC GAGTCGGCAT AA -             #ACCACGGG   3720                                                                  - - CCTGTGATTG CTGGAGTGAT TGGAGCACGC AAGCCTCAGT ATGACATCTG GG -             #GAAACACA   3780                                                                  - - GTCAATGTTG CCAGCCGCAT GGAGAGCACC GGAGAGCTTG GGAAAATCCA GG -             #TTACCGAA   3840                                                                  - - GAGACATGCA CTATCCTCCA GGGACTCGGA TATTCGTGTG AATGCCGTGG GC -             #TGATCAAC   3900                                                                  - - GTCAAAGGCA AAGGGGAACT GCGGACTTAC TTTGTATGTA CAGACACTGC CA -             #AGTTTCAA   3960                                                                  - - GGGCTGGGGC TAAACTGAGG TGGCTGGTGG TCAGCCTCCT TCCCCGAGGG AG -             #CCAAGAAT   4020                                                                  - - GTAGCCCCAT GTCTGTTGCA GTGGCTTCTT TGGACTTGCA CTACAGGATG GC -             #TTTGACCT   4080                                                                  - - GTGCATCAGA TTCTGTTTGA AGCAGCTACT GCGTTGTACA CAGCGGCTCT GT -             #GCTTCAGC   4140                                                                  - - CTCTACAGTT CCAAATTAGC TAGACCACTG GTCTACTACA GGCTGTGTTC AT -             #TTCCAGGG   4200                                                                  - - TGCTGGGGAA GAGACTTCAG TGCATGACCA AGATAGACAT CCACCTTGGT GC -             #CAGTGAAC   4260                                                                  - - AGCATTCACA GGAGACAAAA GCTCTACTGG CTACAGGAGG CTCAGCCAGG CT -             #TATTAGCA   4320                                                                  - - TGGGTTGCTG CTTGCCTTCC TCCCATCAAA TCTCCCATGG GATGTTATTC TT -             #TCAATTAG   4380                                                                  - - GCATTCTGGT AAATGGAGTT GAAAACTGTG TATATTGGTG GGTAGTCTCA AA -             #ACAGCAGA   4440                                                                  - - GAAAATGTCT GATCTACACT TGTCACTTTT TTCCATCTCT GGCTTATGTT TG -             #AAACGGAC   4500                                                                  - - ATGTCATAAA CAGAGTTTTA GCTTTACCAC TGACTCTTAG ATGCTAGACA AA -             #GATCTCCA   4560                                                                  - - CCTTTCTAGT GTATTTTCTC TTGTTAACCA CAGACTACAA GTAAATAGGT CT -             #GCTGTCTA   4620                                                                  - - GTGTCCTTTT ATAGGATCAG ATTGGCTGCA GGGACAGAGT TCTAAGGAAT GG -             #GGCTCATA   4680                                                                  - - GCAGCAGCAA AGCTTTGAAT TTGCAATCAA GCATTTTTTG ATGCAAGTCT TT -             #TGGGACAA   4740                                                                  - - GGCTCAGGAA GTTTAAAGTC TAGAAATGAG GTATGATGTT TCAGTTTTTC TG -             #TGTGTACT   4800                                                                  - - TTATTTATTT TGGAGTCAGG GTCAGCCTAG CTTGGTCTCA AACTTCTAAC CT -             #CCTGTTTC   4860                                                                  - - GGTCTCCGGA GTGTTTCAAT TACAGATGTG CACGACTATC TCCAGCTGTT TC -             #TGTGGGAA   4920                                                                  - - AGCCTGTGTA GACAGGCTTG GACAACTTTG TAGCACTTGC CTTTTCTCCA GT -             #CTTCTGAG   4980                                                                  - - CTGACGACAG AGCTTCAAGA ACAATCCACT TGACAGGAAC ATGTGTTCAG GA -             #CTCTGGCC   5040                                                                  - - TGTGAACTGA GCCCCTCAAC AAATGCCAAA TTGTTCTTAT GCAAATGAGT CA -             #AGGCAAAA   5100                                                                  - - GCCAGCTTCG TGAGATGGGT GTCTTACTGT GCTTAGCTCC AGAGCATTCC GA -             #GAGAGATG   5160                                                                  - - ACCAAACACC CCACTCCTTT TTGGAAATGA CCTCGTGCC      - #                       - #  5199                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1099 amino - #acids                                                (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                               - - Met Pro Ala Lys Gly Arg Tyr Phe Leu Asn Gl - #u Gly Asp Glu Gly Pro       1               5   - #                10  - #                15                - - Asp Gln Ala Ala Leu Tyr Glu Lys Tyr Arg Le - #u Thr Ser Leu His Gly                   20      - #            25      - #            30                    - - Pro Leu Leu Leu Leu Leu Leu Leu Val Ala Al - #a Ala Thr Cys Ile Ala               35          - #        40          - #        45                        - - Leu Ile Ser Ile Ala Phe Ser His Glu Asp Le - #u Arg Arg His Gln Val           50              - #    55              - #    60                            - - Val Leu Gly Thr Ala Phe Leu Met Leu Thr Le - #u Phe Val Ala Leu Tyr       65                  - #70                  - #75                  - #80         - - Val Leu Val Tyr Val Glu Cys Leu Val Gln Ar - #g Trp Leu Arg Ala Leu                       85  - #                90  - #                95                - - Ala Leu Leu Thr Trp Ala Cys Leu Met Val Le - #u Gly Ser Val Leu Met                   100      - #           105      - #           110                   - - Trp Asp Ser Leu Glu Asn Glu Ala His Ala Tr - #p Glu Gln Val Pro Phe               115          - #       120          - #       125                       - - Phe Leu Phe Val Val Phe Val Val Tyr Ala Le - #u Leu Pro Leu Ser Arg           130              - #   135              - #   140                           - - Arg Ala Ala Ile Val Val Gly Val Thr Ser Th - #r Val Ser His Leu Leu       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Val Phe Gly Ala Val Thr Arg Ala Phe Gln Th - #r Ser Met Ser Ser         Thr                                                                                              165  - #               170  - #               175              - - Gln Leu Gly Leu Gln Leu Leu Ala Asn Ala Va - #l Ile Leu Leu Gly Gly                   180      - #           185      - #           190                   - - Asn Phe Thr Gly Ala Phe His Lys His Gln Le - #u Gln Asp Ala Ser Arg               195          - #       200          - #       205                       - - Asp Leu Phe Ile Tyr Thr Val Lys Cys Ile Gl - #n Ile Arg Arg Lys Leu           210              - #   215              - #   220                           - - Arg Val Glu Lys Arg Gln Gln Glu Asn Leu Le - #u Leu Ser Val Leu Pro       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Ala His Ile Ser Met Gly Met Lys Leu Ala Il - #e Ile Glu Arg Leu         Lys                                                                                              245  - #               250  - #               255              - - Glu Gly Gly Asp Arg His Tyr Met Pro Asp As - #n Asn Phe His Ser Leu                   260      - #           265      - #           270                   - - Tyr Val Lys Arg His Gln Asn Val Ser Ile Le - #u Tyr Ala Asp Ile Val               275          - #       280          - #       285                       - - Gly Phe Thr Arg Leu Ala Ser Asp Cys Ser Pr - #o Lys Glu Leu Val Val           290              - #   295              - #   300                           - - Val Leu Asn Glu Leu Phe Gly Lys Phe Asp Gl - #n Ile Ala Lys Ala Asn       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Glu Cys Met Arg Ile Lys Ile Leu Gly Asp Cy - #s Tyr Tyr Cys Val         Ser                                                                                              325  - #               330  - #               335              - - Gly Leu Pro Val Ser Leu Pro Thr His Ala Ar - #g Asn Cys Val Lys Met                   340      - #           345      - #           350                   - - Gly Leu Asp Ile Cys Glu Ala Ile Lys Gln Va - #l Arg Glu Ala Thr Gly               355          - #       360          - #       365                       - - Val Asp Ile Ser Met Arg Val Gly Ile His Se - #r Gly Asn Val Leu Cys           370              - #   375              - #   380                           - - Gly Val Ile Gly Leu Arg Lys Trp Gln Tyr As - #p Val Trp Ser His Asp       385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - Val Ser Leu Ala Asn Arg Met Glu Ala Ala Gl - #y Val Pro Gly Arg         Val                                                                                              405  - #               410  - #               415              - - His Ile Thr Glu Ala Thr Leu Asn His Leu As - #p Lys Ala Tyr Glu Val                   420      - #           425      - #           430                   - - Glu Asp Gly His Gly Glu Gln Arg Asp Pro Ty - #r Leu Lys Glu Met Asn               435          - #       440          - #       445                       - - Ile Arg Thr Tyr Leu Val Ile Asp Pro Arg Se - #r Gln Gln Pro Pro Pro           450              - #   455              - #   460                           - - Pro Ser His His Leu Ser Lys Pro Lys Gly As - #p Ala Thr Leu Lys Met       465                 4 - #70                 4 - #75                 4 -       #80                                                                               - - Arg Ala Ser Val Arg Val Thr Arg Tyr Leu Gl - #u Ser Trp Gly Ala         Ala                                                                                              485  - #               490  - #               495              - - Arg Pro Phe Ala His Leu Asn His Arg Glu Se - #r Val Ser Ser Ser Glu                   500      - #           505      - #           510                   - - Thr Pro Ile Ser Asn Gly Arg Arg Gln Lys Al - #a Ile Pro Leu Arg Arg               515          - #       520          - #       525                       - - His Arg Ala Pro Asp Arg Ser Ala Ser Pro Ly - #s Gly Arg Leu Glu Asp           530              - #   535              - #   540                           - - Asp Cys Asp Asp Glu Met Leu Ser Ala Ile Gl - #u Gly Leu Ser Ser Thr       545                 5 - #50                 5 - #55                 5 -       #60                                                                               - - Arg Pro Cys Cys Ser Lys Ser Asp Asp Phe Hi - #s Thr Phe Gly Pro         Ile                                                                                              565  - #               570  - #               575              - - Phe Leu Glu Lys Gly Phe Glu Arg Glu Tyr Ar - #g Leu Val Pro Ile Pro                   580      - #           585      - #           590                   - - Arg Ala Arg Tyr Asp Phe Ala Cys Ala Ser Le - #u Val Phe Val Cys Ile               595          - #       600          - #       605                       - - Leu Leu Val His Leu Leu Val Met Pro Arg Me - #t Ala Thr Leu Gly Val           610              - #   615              - #   620                           - - Ser Phe Gly Leu Val Ala Cys Leu Leu Gly Le - #u Val Leu Ser Phe Cys       625                 6 - #30                 6 - #35                 6 -       #40                                                                               - - Phe Ala Thr Glu Phe Ser Arg Cys Phe Pro Se - #r Arg Ser Thr Leu         Gln                                                                                              645  - #               650  - #               655              - - Ala Ile Ser Glu Ser Val Glu Thr Gln Pro Le - #u Val Arg Leu Val Leu                   660      - #           665      - #           670                   - - Val Val Leu Thr Val Gly Ser Leu Leu Thr Va - #l Ala Ile Ile Asn Met               675          - #       680          - #       685                       - - Pro Leu Thr Leu Asn Pro Gly Pro Glu Gln Pr - #o Gly Asp Asn Lys Thr           690              - #   695              - #   700                           - - Ser Pro Leu Ala Ala Gln Asn Arg Val Gly Th - #r Pro Tyr Glu Leu Leu       705                 7 - #10                 7 - #15                 7 -       #20                                                                               - - Pro Tyr Tyr Thr Cys Ser Cys Ile Leu Gly Ph - #e Ile Ala Cys Ser         Val                                                                                              725  - #               730  - #               735              - - Phe Leu Arg Met Ser Leu Glu Leu Lys Ala Me - #t Leu Leu Thr Val Ala                   740      - #           745      - #           750                   - - Leu Val Ala Tyr Leu Leu Leu Phe Asn Leu Se - #r Pro Cys Trp His Val               755          - #       760          - #       765                       - - Ser Gly Asn Ser Thr Glu Thr Asn Gly Thr Gl - #n Arg Thr Arg Leu Leu           770              - #   775              - #   780                           - - Leu Ser Asp Ala Gln Ser Met Pro Ser His Th - #r Leu Ala Pro Gly Ala       785                 7 - #90                 7 - #95                 8 -       #00                                                                               - - Gln Glu Thr Ala Pro Ser Pro Ser Tyr Leu Gl - #u Arg Asp Leu Lys         Ile                                                                                              805  - #               810  - #               815              - - Met Val Asn Phe Tyr Leu Ile Leu Phe Tyr Al - #a Thr Leu Ile Leu Leu                   820      - #           825      - #           830                   - - Ser Arg Gln Ile Asp Tyr Tyr Cys Arg Leu As - #p Cys Leu Trp Lys Lys               835          - #       840          - #       845                       - - Lys Phe Lys Lys Glu His Glu Glu Phe Glu Th - #r Met Glu Asn Val Asn           850              - #   855              - #   860                           - - Arg Leu Leu Leu Glu Asn Val Leu Pro Ala Hi - #s Val Ala Ala His Phe       865                 8 - #70                 8 - #75                 8 -       #80                                                                               - - Ile Gly Asp Lys Ala Ala Glu Asp Trp Tyr Hi - #s Gln Ser Tyr Asp         Cys                                                                                              885  - #               890  - #               895              - - Val Cys Val Met Phe Ala Ser Val Pro Asp Ph - #e Lys Val Phe Tyr Thr                   900      - #           905      - #           910                   - - Glu Cys Asp Val Asn Lys Glu Gly Leu Glu Cy - #s Leu Arg Leu Leu Asn               915          - #       920          - #       925                       - - Glu Ile Ile Ala Asp Phe Asp Glu Leu Leu Le - #u Lys Pro Lys Phe Ser           930              - #   935              - #   940                           - - Gly Val Glu Lys Ile Lys Thr Ile Gly Ser Th - #r Tyr Met Ala Ala Ala       945                 9 - #50                 9 - #55                 9 -       #60                                                                               - - Gly Leu Ser Ala Pro Ser Gly His Glu Asn Gl - #n Asp Leu Glu Arg         Lys                                                                                              965  - #               970  - #               975              - - His Val His Ile Gly Val Leu Val Glu Phe Se - #r Met Ala Leu Met Ser                   980      - #           985      - #           990                   - - Lys Leu Asp Gly Ile Asn Arg His Ser Phe As - #n Ser Phe Arg Leu Arg               995          - #       1000          - #      1005                      - - Val Gly Ile Asn His Gly Pro Val Ile Ala Gl - #y Val Ile Gly Ala Arg           1010             - #   1015              - #  1020                          - - Lys Pro Gln Tyr Asp Ile Trp Gly Asn Thr Va - #l Asn Val Ala Ser Arg       1025                1030 - #                1035 - #               1040         - - Met Glu Ser Thr Gly Glu Leu Gly Lys Ile Gl - #n Val Thr Glu Glu Thr                       1045 - #               1050  - #              1055              - - Cys Thr Ile Leu Gln Gly Leu Gly Tyr Ser Cy - #s Glu Cys Arg Gly Leu                   1060     - #           1065      - #          1070                  - - Ile Asn Val Lys Gly Lys Gly Glu Leu Arg Th - #r Tyr Phe Val Cys Thr               1075         - #       1080          - #      1085                      - - Asp Thr Ala Lys Phe Gln Gly Leu Gly Leu As - #n                               1090             - #   1095                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 4601 base - #pairs                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                               - - CCAGAACTCG CGCTGTCAGT ATTTGCTCTG GAAGTGGCCA CAACCAGGAA GG -              #AACCGAAG     60                                                                  - - AGAGAGCTTC ATCCAGCACA CCTCCAAATT AACTGTTTGG TGACTTGTTA GT -             #GTTAAGGA    120                                                                  - - TACCACCTTT CTCCTGCCCT CCCGCGGGGC GCCCTGCCAC CTCACCATCA CC -             #CACACCTC    180                                                                  - - CTGCCTCATC GCGCCCGCCT CCAGCTCCTC CCCGACTCCC CATGCCGGCG CA -             #ATGGAGTT    240                                                                  - - ATCCGAAGGG CGATGATTGC AGCCACAGCT GCTAACTTAG CACCCATCCT AG -             #CCATCGGT    300                                                                  - - CACCCTTGGC CAGCCTTTCT GCAGCCGCCG AGACGTGGGC GTCCAGAGTC CA -             #GTGCCGCA    360                                                                  - - GCCAGGACCC GCCCGACGCG CAGCAGAAGC GCGGCGCCCA GGCGCCTCTT AG -             #AGAAGACT    420                                                                  - - GCGCCAAGAC GCTGAGCCTA GAGCCAGCCC AGGAGCTTGA GCGCAGAAAG GG -             #ACTGCGTG    480                                                                  - - GCTCAGAGTG CCGAGGATTT CGTGGACAAA TAGCCACCAC TTTCACTGCG TA -             #TCCCAACG    540                                                                  - - GAGCTGATTG GCGGGAAGGT GGCTTGCCAG TACGCAGGGG TCCACTGCAA TC -             #TGCCAGGA    600                                                                  - - GCCCCTTGCT CTCGGGCTCA CTATGACTTG ACCTGTGGGG GCCCTGCTCA GG -             #GCTCCTGC    660                                                                  - - AATCGAGCAG CCTAGGAAAG AGGGGATCGC TGCCAAGCGG CCTTCGGGTC TC -             #TAAAAACC    720                                                                  - - CCTACCCGCA ACTGCCACAA CCAGTGTCTG AGCCTCCCTC CCTGCGGACC CC -             #AGCCATGG    780                                                                  - - AACTCTCGGA TGTGCACTGC CTTAGCGGCA GCGAGGAACT CTATACCATT CA -             #CCCGACGC    840                                                                  - - CCCCGGCCGC GGACGGCGGG AGCGGCTCTC GGCCGCAGCG GCTGCTGTGG CA -             #GACGGCGG    900                                                                  - - TGCGGCACAT CACCGAGCAG CGCTTCATCC ACGGCCACCG AGGCGGCGGC GG -             #CGGGGGCT    960                                                                  - - CCCGCAAAGC CTCGAACCCT GCGGGCAGCG GACCAAACCA CCACGCGCCG CA -             #GCTGTCTA   1020                                                                  - - GCGACTCGGT GCTGCCTCTC TATTCTCTGG GCTCCGGAGA GCGAGCGCAC AA -             #CACCGGTG   1080                                                                  - - GCACCAAAGT CTTTCCGGAA CGCAGCGGGA GCGGCAGTGC CAGTGGCAGC GG -             #GGGTGGGG   1140                                                                  - - GCGACTTGGG CTTCCTGCAC CTTGACTGTG CCCCAAGTAA CTCGGATTTC TT -             #CCTCAATG   1200                                                                  - - GGGGATACAG CTACCGTGGG GTCATTTTCC CAACCCTACG CAACTCCTTC AA -             #GTCTCGGG   1260                                                                  - - ATCTGGAGCG CCTCTATCAA CGCTATTTCC TGGGCCAGAG GCGCAAATCG GA -             #GGTAGTGA   1320                                                                  - - TGAACGTGCT GGATGTACTA ACCAAACTCA CCCTTCTAGT CCTTCACTTG AG -             #CCTAGCCT   1380                                                                  - - CGGCTCCCAT GGACCCTCTC AAGGGCATCC TGCTAGGCTT TTTCACTGGC AT -             #CGAGGTGG   1440                                                                  - - TGATCTGCGC CCTCGTGGTG GTCAGGAAGG ACACCACCTC CCACACTTAC CT -             #GCAATACA   1500                                                                  - - GCGGCGTGGT CACTTGGGTG GCTATGACCA CCCAGATTCT GGCAGCAGGC CT -             #GGGCTATG   1560                                                                  - - GGCTTCTGGG CGACGGCATA GGCTACGTGC TTTTTACACT CTTCGCCACC TA -             #CAGCATGC   1620                                                                  - - TTCCGCTGCC TCTCACCTGG GCCATCTTGG CCGGCCTGGG CACATCCTTG CT -             #GCAAGTCA   1680                                                                  - - CACTTCAAGT GCTCATACCC AGACTAGCGG TCTTTTCCAT CAACCAGGTC CT -             #GGCCCAGG   1740                                                                  - - TGGTGCTCTT CATGTGCATG AATACAGCAG GCATCTTCAT CAGTTACCTT TC -             #AGACCGCG   1800                                                                  - - CCCAGCGGCA GGCCTTCCTG GAGACCCGGA GGTGTGTGGA GGCCAGGCTC CG -             #CCTGGAGA   1860                                                                  - - CAGAGAACCA AAGACAGGAG CGGCTTGTGC TCTCTGTGCT CCCCAGGTTT GT -             #CGTCCTAG   1920                                                                  - - AAATGATCAA TGACATGACC AATGTGGAGG ACGAGCACCT GCAGCATCAG TT -             #CCACCGCA   1980                                                                  - - TCTACATCCA TCGCTACGAG AACGTCAGTA TTCTTTTTGC AGATGTCAAA GG -             #ATTTACCA   2040                                                                  - - ACCTCTCTAC GACCTTGTCT GCTCAGGAGC TTGTCAGGAT GCTCAACGAG CT -             #CTTTGCCA   2100                                                                  - - GATTTGATCG GCTGGCCCAT GAGCATCACT GTCTTCGCAT TAAAATCCTG GG -             #GGACTGCT   2160                                                                  - - ACTACTGTGT GTCAGGACTG CCTGAGCCCC GCCAGGACCA TGCTCATTGC TG -             #TGTTGAAA   2220                                                                  - - TGGGCCTCAG CATGATCAAA ACTATCAGGT TTGTGAGGTC CAGAACGAAG CA -             #TGATGTTG   2280                                                                  - - ACATGCGAAT TGGAATCCAT TCAGGCTCTG TGCTGTGTGG TGTGTTGGGC CT -             #GAGAAAAT   2340                                                                  - - GGCAGTTTGA TGTCTGGTCT TGGGATGTGG ACATCGCAAA CAAACTTGAA TC -             #TGGAGGAA   2400                                                                  - - TCCCTGGGAG AATTCACATT TCCAAAGCCA CACTGGATTG CCTCAGTGGT GA -             #CTATAATG   2460                                                                  - - TGGAAGAGGG CCACGGTAAG GAGAGGAATG AATTCTTGAG GAAGCATAAT AT -             #AGAGACCT   2520                                                                  - - ATTTGATTAA GCAGCCCGAG GAGAGTTTGC TATCCTTGCC TGAAGATATA GT -             #TAAGGAGT   2580                                                                  - - CGGTGAGCTG CTCGGACAGG AGAAACAGTG GGGCAACGTT CACAGAAGGA TC -             #CTGGAGCC   2640                                                                  - - CAGAACTGCC ATTCGACAAC ATCGTGGGCA AACAGAATAC TCTGGCTGCC CT -             #AACAAGAA   2700                                                                  - - ATTCAATAAA TCTGCTTCCA AACCATCTCG CACAAGCTTT GCATGTCCAG TC -             #TGGGCCTG   2760                                                                  - - AGGAAATTAA CAAGAGAATA GAGCATACCA TCGACTTGCG GAGTGGCGAT AA -             #GTTGAGAA   2820                                                                  - - GAGAGCATAT CAAGCCATTC TCACTGATGT TTAAAGACTC CAGCCTGGAG CA -             #CAAGTATT   2880                                                                  - - CTCAAATGCG GGATGAAGTA TTCAAGTCAA ACTTGGTCTG TGCATTTATC GT -             #TCTTCTGT   2940                                                                  - - TTATCACTGC GATTCAAAGT TTGCTTCCTT CTTCGAGGCT GATGCCTATG AC -             #CATCCAGT   3000                                                                  - - TCTCCATCCT GATCATGCTG CACTCCGCCC TGGTCCTCAT CACCACGGCA GA -             #AGACTATA   3060                                                                  - - AGTGTCTGCC TCTCATTCTC CGCAAAACCT GTTGTTGGAT TAACGAGACC TA -             #TTTGGCCC   3120                                                                  - - GCAACGTCAT CATCTTTGCT TCCATCTTGA TTAATTTCCT GGGAGCCGTC AT -             #AAATATTC   3180                                                                  - - TGTGGTGTGA TTTTGACAAG TCGATACCCT TGAAGAACCT GACTTTCAAT TC -             #CTCAGCTG   3240                                                                  - - TGTTTACAGA TATCTGCTCC TACCCAGAGT ACTTTGTCTT CACTGGGGTG TT -             #GGCCATGG   3300                                                                  - - TGACGTGTGC AGTATTTCTC CGGCTTAACT CTGTCCTGAA GCTGGCTGTG CT -             #GCTCATTA   3360                                                                  - - TGATCGCCAT CTACGCCCTG CTGACAGAGA CCATCTATGC AGGTCTCTTT CT -             #GAGTTATG   3420                                                                  - - ACAACCTGAA CCACAGTGGA GAAGATTTTC TGGGGACCAA GGAGGCATCA CT -             #GCTACTGA   3480                                                                  - - TGGCCATGTT CCTTCTTGCT GTATTCTACC ATGGACAGCA GCTGGAGTAC AC -             #AGCCCGCC   3540                                                                  - - TAGATTTCCT GTGGCGAGTA CAGGCCAAAG AGGAGATCAA CGAGATGAAG GA -             #CTTGAGGG   3600                                                                  - - AACACAATGA GAACATGCTT CGCAATATCT TACCCGGCCA CGTGGCCCGC CA -             #CTTCCTGG   3660                                                                  - - AGAAAGACAG AGACAATGAG GAGCTGTATT CTCAATCCTA TGATGCCGTT GG -             #GGTAATGT   3720                                                                  - - TTGCCTCCAT TCCTGGATTT GCAGACTTTT ACTCTCAGAC AGAAATGAAC AA -             #CCAGGGAG   3780                                                                  - - TGGAATGTCT GCGCTTGCTG AATGAGATCA TTGCTGACTT TGATGAGTTA CT -             #TGGAGAGG   3840                                                                  - - ACCGCTTTCA GGACATAGAG AAGATTAAGA CCATTGGTAG TACATACATG GC -             #TGTCTCAG   3900                                                                  - - GACTGTCACC AGAGAAACAG CAATGTGAAG ATAAATGGGG ACATTTGTGT GC -             #CCTGGCTG   3960                                                                  - - ACTTCTCTCT TGCCCTGACT GAAAGCATAC AAGAGATCAA CAAGCATTCG TT -             #CAACAATT   4020                                                                  - - TTGAACTCCG TATTGGCATC AGCCATGGCT CAGTGGTAGC AGGTGTAATT GG -             #CGCTAAGA   4080                                                                  - - AACCACAGTA TGACATTTGG GGTAAAACTG TGAACTTGGC AAGCCGAATG GA -             #CAGCACAG   4140                                                                  - - GAGTGAGTGG CCGGATCCAA GTTCCTGAGG AGACCTATCT CATCCTGAAG GA -             #TCAGGGCT   4200                                                                  - - TTGCCTTCGA CTACCGGGGA GAGATATATG TGAAGGGCAT CAGCGAACAA GA -             #AGGGAAAA   4260                                                                  - - TCAAAACATA TTTTCTCCTG GGACGAGTCC AACCCAACCC ATTCATCTTA CC -             #CCCAAGAA   4320                                                                  - - GACTTCCTGG GCAGTACTCT CTGGCTGCAG TTGTCCTTGG GCTTGTCCAG TC -             #TCTCAACA   4380                                                                  - - GGCAAAGGCA GAAGCAACTA CTCAATGAGA ACAGCAACTC TGGCATCATC AA -             #GAGCCATT   4440                                                                  - - ACAACCGGCG GACTTTGCTA ACGCCAAGTG GGCCAGAGCC TGGAGCACAG GC -             #TGAAGGCA   4500                                                                  - - CTGACAAATC CGATTTGCCA TAAAAGCATT TTCTTTGTGT TTCTTTCTCT TT -             #TTTTGTAT   4560                                                                  - - TTCTTTTATA TATAAAATAA ATATACTAAT AAAAAGGTTT G    - #                       - # 4601                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1248 amino - #acids                                                (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                               - - Met Glu Leu Ser Asp Val His Cys Leu Ser Gl - #y Ser Glu Glu Leu Tyr       1               5   - #                10  - #                15                - - Thr Ile His Pro Thr Pro Pro Ala Ala Asp Gl - #y Gly Ser Gly Ser Arg                   20      - #            25      - #            30                    - - Pro Gln Arg Leu Leu Trp Gln Thr Ala Val Ar - #g His Ile Thr Glu Gln               35          - #        40          - #        45                        - - Arg Phe Ile His Gly His Arg Gly Gly Gly Gl - #y Gly Gly Ser Arg Lys           50              - #    55              - #    60                            - - Ala Ser Asn Pro Ala Gly Ser Gly Pro Asn Hi - #s His Ala Pro Gln Leu       65                  - #70                  - #75                  - #80         - - Ser Ser Asp Ser Val Leu Pro Leu Tyr Ser Le - #u Gly Ser Gly Glu Arg                       85  - #                90  - #                95                - - Ala His Asn Thr Gly Gly Thr Lys Val Phe Pr - #o Glu Arg Ser Gly Ser                   100      - #           105      - #           110                   - - Gly Ser Ala Ser Gly Ser Gly Gly Gly Gly As - #p Leu Gly Phe Leu His               115          - #       120          - #       125                       - - Leu Asp Cys Ala Pro Ser Asn Ser Asp Phe Ph - #e Leu Asn Gly Gly Tyr           130              - #   135              - #   140                           - - Ser Tyr Arg Gly Val Ile Phe Pro Thr Leu Ar - #g Asn Ser Phe Lys Ser       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Arg Asp Leu Glu Arg Leu Tyr Gln Arg Tyr Ph - #e Leu Gly Gln Arg         Arg                                                                                              165  - #               170  - #               175              - - Lys Ser Glu Val Val Met Asn Val Leu Asp Va - #l Leu Thr Lys Leu Thr                   180      - #           185      - #           190                   - - Leu Leu Val Leu His Leu Ser Leu Ala Ser Al - #a Pro Met Asp Pro Leu               195          - #       200          - #       205                       - - Lys Gly Ile Leu Leu Gly Phe Phe Thr Gly Il - #e Glu Val Val Ile Cys           210              - #   215              - #   220                           - - Ala Leu Val Val Val Arg Lys Asp Thr Thr Se - #r His Thr Tyr Leu Gln       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Tyr Ser Gly Val Val Thr Trp Val Ala Met Th - #r Thr Gln Ile Leu         Ala                                                                                              245  - #               250  - #               255              - - Ala Gly Leu Gly Tyr Gly Leu Leu Gly Asp Gl - #y Ile Gly Tyr Val Leu                   260      - #           265      - #           270                   - - Phe Thr Leu Phe Ala Thr Tyr Ser Met Leu Pr - #o Leu Pro Leu Thr Trp               275          - #       280          - #       285                       - - Ala Ile Leu Ala Gly Leu Gly Thr Ser Leu Le - #u Gln Val Thr Leu Gln           290              - #   295              - #   300                           - - Val Leu Ile Pro Arg Leu Ala Val Phe Ser Il - #e Asn Gln Val Leu Ala       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Gln Val Val Leu Phe Met Cys Met Asn Thr Al - #a Gly Ile Phe Ile         Ser                                                                                              325  - #               330  - #               335              - - Tyr Leu Ser Asp Arg Ala Gln Arg Gln Ala Ph - #e Leu Glu Thr Arg Arg                   340      - #           345      - #           350                   - - Cys Val Glu Ala Arg Leu Arg Leu Glu Thr Gl - #u Asn Gln Arg Gln Glu               355          - #       360          - #       365                       - - Arg Leu Val Leu Ser Val Leu Pro Arg Phe Va - #l Val Leu Glu Met Ile           370              - #   375              - #   380                           - - Asn Asp Met Thr Asn Val Glu Asp Glu His Le - #u Gln His Gln Phe His       385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - Arg Ile Tyr Ile His Arg Tyr Glu Asn Val Se - #r Ile Leu Phe Ala         Asp                                                                                              405  - #               410  - #               415              - - Val Lys Gly Phe Thr Asn Leu Ser Thr Thr Le - #u Ser Ala Gln Glu Leu                   420      - #           425      - #           430                   - - Val Arg Met Leu Asn Glu Leu Phe Ala Arg Ph - #e Asp Arg Leu Ala His               435          - #       440          - #       445                       - - Glu His His Cys Leu Arg Ile Lys Ile Leu Gl - #y Asp Cys Tyr Tyr Cys           450              - #   455              - #   460                           - - Val Ser Gly Leu Pro Glu Pro Arg Gln Asp Hi - #s Ala His Cys Cys Val       465                 4 - #70                 4 - #75                 4 -       #80                                                                               - - Glu Met Gly Leu Ser Met Ile Lys Thr Ile Ar - #g Phe Val Arg Ser         Arg                                                                                              485  - #               490  - #               495              - - Thr Lys His Asp Val Asp Met Arg Ile Gly Il - #e His Ser Gly Ser Val                   500      - #           505      - #           510                   - - Leu Cys Gly Val Leu Gly Leu Arg Lys Trp Gl - #n Phe Asp Val Trp Ser               515          - #       520          - #       525                       - - Trp Asp Val Asp Ile Ala Asn Lys Leu Glu Se - #r Gly Gly Ile Pro Gly           530              - #   535              - #   540                           - - Arg Ile His Ile Ser Lys Ala Thr Leu Asp Cy - #s Leu Ser Gly Asp Tyr       545                 5 - #50                 5 - #55                 5 -       #60                                                                               - - Asn Val Glu Glu Gly His Gly Lys Glu Arg As - #n Glu Phe Leu Arg         Lys                                                                                              565  - #               570  - #               575              - - His Asn Ile Glu Thr Tyr Leu Ile Lys Gln Pr - #o Glu Glu Ser Leu Leu                   580      - #           585      - #           590                   - - Ser Leu Pro Glu Asp Ile Val Lys Glu Ser Va - #l Ser Cys Ser Asp Arg               595          - #       600          - #       605                       - - Arg Asn Ser Gly Ala Thr Phe Thr Glu Gly Se - #r Trp Ser Pro Glu Leu           610              - #   615              - #   620                           - - Pro Phe Asp Asn Ile Val Gly Lys Gln Asn Th - #r Leu Ala Ala Leu Thr       625                 6 - #30                 6 - #35                 6 -       #40                                                                               - - Arg Asn Ser Ile Asn Leu Leu Pro Asn His Le - #u Ala Gln Ala Leu         His                                                                                              645  - #               650  - #               655              - - Val Gln Ser Gly Pro Glu Glu Ile Asn Lys Ar - #g Ile Glu His Thr Ile                   660      - #           665      - #           670                   - - Asp Leu Arg Ser Gly Asp Lys Leu Arg Arg Gl - #u His Ile Lys Pro Phe               675          - #       680          - #       685                       - - Ser Leu Met Phe Lys Asp Ser Ser Leu Glu Hi - #s Lys Tyr Ser Gln Met           690              - #   695              - #   700                           - - Arg Asp Glu Val Phe Lys Ser Asn Leu Val Cy - #s Ala Phe Ile Val Leu       705                 7 - #10                 7 - #15                 7 -       #20                                                                               - - Leu Phe Ile Thr Ala Ile Gln Ser Leu Leu Pr - #o Ser Ser Arg Leu         Met                                                                                              725  - #               730  - #               735              - - Pro Met Thr Ile Gln Phe Ser Ile Leu Ile Me - #t Leu His Ser Ala Leu                   740      - #           745      - #           750                   - - Val Leu Ile Thr Thr Ala Glu Asp Tyr Lys Cy - #s Leu Pro Leu Ile Leu               755          - #       760          - #       765                       - - Arg Lys Thr Cys Cys Trp Ile Asn Glu Thr Ty - #r Leu Ala Arg Asn Val           770              - #   775              - #   780                           - - Ile Ile Phe Ala Ser Ile Leu Ile Asn Phe Le - #u Gly Ala Val Ile Asn       785                 7 - #90                 7 - #95                 8 -       #00                                                                               - - Ile Leu Trp Cys Asp Phe Asp Lys Ser Ile Pr - #o Leu Lys Asn Leu         Thr                                                                                              805  - #               810  - #               815              - - Phe Asn Ser Ser Ala Val Phe Thr Asp Ile Cy - #s Ser Tyr Pro Glu Tyr                   820      - #           825      - #           830                   - - Phe Val Phe Thr Gly Val Leu Ala Met Val Th - #r Cys Ala Val Phe Leu               835          - #       840          - #       845                       - - Arg Leu Asn Ser Val Leu Lys Leu Ala Val Le - #u Leu Ile Met Ile Ala           850              - #   855              - #   860                           - - Ile Tyr Ala Leu Leu Thr Glu Thr Ile Tyr Al - #a Gly Leu Phe Leu Ser       865                 8 - #70                 8 - #75                 8 -       #80                                                                               - - Tyr Asp Asn Leu Asn His Ser Gly Glu Asp Ph - #e Leu Gly Thr Lys         Glu                                                                                              885  - #               890  - #               895              - - Ala Ser Leu Leu Leu Met Ala Met Phe Leu Le - #u Ala Val Phe Tyr His                   900      - #           905      - #           910                   - - Gly Gln Gln Leu Glu Tyr Thr Ala Arg Leu As - #p Phe Leu Trp Arg Val               915          - #       920          - #       925                       - - Gln Ala Lys Glu Glu Ile Asn Glu Met Lys As - #p Leu Arg Glu His Asn           930              - #   935              - #   940                           - - Glu Asn Met Leu Arg Asn Ile Leu Pro Gly Hi - #s Val Ala Arg His Phe       945                 9 - #50                 9 - #55                 9 -       #60                                                                               - - Leu Glu Lys Asp Arg Asp Asn Glu Glu Leu Ty - #r Ser Gln Ser Tyr         Asp                                                                                              965  - #               970  - #               975              - - Ala Val Gly Val Met Phe Ala Ser Ile Pro Gl - #y Phe Ala Asp Phe Tyr                   980      - #           985      - #           990                   - - Ser Gln Thr Glu Met Asn Asn Gln Gly Val Gl - #u Cys Leu Arg Leu Leu               995          - #       1000          - #      1005                      - - Asn Glu Ile Ile Ala Asp Phe Asp Glu Leu Le - #u Gly Glu Asp Arg Phe           1010             - #   1015              - #  1020                          - - Gln Asp Ile Glu Lys Ile Lys Thr Ile Gly Se - #r Thr Tyr Met Ala Val       1025                1030 - #                1035 - #               1040         - - Ser Gly Leu Ser Pro Glu Lys Gln Gln Cys Gl - #u Asp Lys Trp Gly His                       1045 - #               1050  - #              1055              - - Leu Cys Ala Leu Ala Asp Phe Ser Leu Ala Le - #u Thr Glu Ser Ile Gln                   1060     - #           1065      - #          1070                  - - Glu Ile Asn Lys His Ser Phe Asn Asn Phe Gl - #u Leu Arg Ile Gly Ile               1075         - #       1080          - #      1085                      - - Ser His Gly Ser Val Val Ala Gly Val Ile Gl - #y Ala Lys Lys Pro Gln           1090             - #   1095              - #  1100                          - - Tyr Asp Ile Trp Gly Lys Thr Val Asn Leu Al - #a Ser Arg Met Asp Ser       1105                1110 - #                1115 - #               1120         - - Thr Gly Val Ser Gly Arg Ile Gln Val Pro Gl - #u Glu Thr Tyr Leu Ile                       1125 - #               1130  - #              1135              - - Leu Lys Asp Gln Gly Phe Ala Phe Asp Tyr Ar - #g Gly Glu Ile Tyr Val                   1140     - #           1145      - #          1150                  - - Lys Gly Ile Ser Glu Gln Glu Gly Lys Ile Ly - #s Thr Tyr Phe Leu Leu               1155         - #       1160          - #      1165                      - - Gly Arg Val Gln Pro Asn Pro Phe Ile Leu Pr - #o Pro Arg Arg Leu Pro           1170             - #   1175              - #  1180                          - - Gly Gln Tyr Ser Leu Ala Ala Val Val Leu Gl - #y Leu Val Gln Ser Leu       1185                1190 - #                1195 - #               1200         - - Asn Arg Gln Arg Gln Lys Gln Leu Leu Asn Gl - #u Asn Ser Asn Ser Gly                       1205 - #               1210  - #              1215              - - Ile Ile Lys Ser His Tyr Asn Arg Arg Thr Le - #u Leu Thr Pro Ser Gly                   1220     - #           1225      - #          1230                  - - Pro Glu Pro Gly Ala Gln Ala Glu Gly Thr As - #p Lys Ser Asp Leu Pro               1235         - #       1240          - #      1245                      - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1652 base - #pairs                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                               - - ACCATGGCTG AGCGCGCCCA GAGGAAGGCC TTCCTGCAGG CCCGGAACTG CA -              #TTGAGGAC     60                                                                  - - CGGCTGAGGC TGGAGGATGA GAATGAGAAG CAGGAGCGGC TGCTCATGAG CC -             #TCCTGCCT    120                                                                  - - CGGAATGTTG CTATGGAGAT GAAGGAGGAC TTCCTGAAGC CCCCTGAGAG GA -             #TTTTCCAC    180                                                                  - - AAGATTTACA TCCAGCGGCA TGACAACGTG AGCATCCTCT TTGCAGACAT CG -             #TGGGCTTC    240                                                                  - - ACAGGCTTGG CGTCACAGTG CACGGCCCAG GAGCTGGTGA AACTCCTCAA TG -             #AGCTCTTC    300                                                                  - - GGGAAGTTTG ACGAGCTGGC CACAGAGAAC CACTGCCGCC GCATCAAGAT CC -             #TGGGAGAT    360                                                                  - - TGCTACTACT GCGTGTCTGG CCTCACTCAG CCCAAGACTG ACCACGCCCA CT -             #GCTGTGTG    420                                                                  - - GAGATGGGCC TGGACATGAT CGACACCATC ACGTCCGTGG CTGAGGCCAC TG -             #AGGTGGAC    480                                                                  - - TTGAACATGC GTGTGGGGCT GCACACCGGC AGGGTCCTCT GCGGGGTCCT GG -             #GCCTGCGT    540                                                                  - - AAGTGGCAGT ATGATGTGTG GTCCAACGAC GTGACCCTGG CCAACGTCAT GG -             #AGGCTGCC    600                                                                  - - GGCCTGCCTG GGAAGGTTCA CATCACAAAG ACCACCCTGG CGTGCCTGAA TG -             #GTGACTAT    660                                                                  - - GAGGTGGAGC CGGGACACGG ACACGAGAGG AACAGTTTTC TGAAAACTCA TA -             #ACATTGAG    720                                                                  - - ACCTTTTTTA TTGTGCCCTC GCATCGGCGA GCGGCCGCTG GAGGCATGCC TC -             #CAGCGGCC    780                                                                  - - GCTGGAGGCA TGAGACAGAG TGAATATTAC TGTAGGTTAG ACTTCTTGTG GA -             #AGAACAAG    840                                                                  - - TTCAAAAAAG AGCGGGAGGA GATAGAAACC ATGGAGAACC TAAATCGAGT GC -             #TGCTGGAG    900                                                                  - - AACGTGCTTC CTGCACACGT GGCTGAACAC TTCCTGGCCA GGAGCCTGAA AA -             #ATGAGGAG    960                                                                  - - CTGTACCACC AGTCCTACGA CTGTGTCTGT GTCATGTTTG CCTCCATTCC GG -             #ACTTCAAG   1020                                                                  - - GAGTTCTACA CAGAGTCAGA TGTGAACAAG GAAGGCTTGG AATGCCTGCG GC -             #TCCTGAAT   1080                                                                  - - GAGATCATTG CTGACTTTGA TGATCTGCTT TCTAAGCCAA AGTTCAGTGG TG -             #TTGAAAAG   1140                                                                  - - ATCAAGACCA TTGGGAGCAC ATACATGGCA GCCACGGGAC TGAGTGCCAT AC -             #CCAGCCAG   1200                                                                  - - GAGCACGCCC AGGAACCTGA GCGTCAGTAC ATGCACATAG GCACCATGGT GG -             #AGTTTGCA   1260                                                                  - - TATGCCCTGG TGGGAAAACT GGATGCCATC AATAAGCACT CCTTCAACGA CT -             #TCAAACTG   1320                                                                  - - CGAGTGGGTA TCAACCATGG GCCTGTAATA GCTGGCGTCA TAGGGGCTCA AA -             #AGCCACAG   1380                                                                  - - TATGACATCT GGGGCAACAC TGTCAACGTG GCCAGCAGAA TGGACAGCAC CG -             #GGGTCCTG   1440                                                                  - - GACAAAATAC AGGTGACTGA GGAGACAAGC CTCATCTTGC AGACGCTTGG CT -             #ACACGTGT   1500                                                                  - - ACATGTCGAG GTATCATCAA TGTGAAGGGG AAAGGGGACC TGAAGACATA TT -             #TTGTAAAC   1560                                                                  - - ACAGAGATGT CAAGGTCCCT TTCTCAGAGC AACTTGGCAT CCTGAGAAGC TG -             #TCTCTTCC   1620                                                                  - - TGACAAGAAG AATGTACTTG CAGGAAGGTA CC       - #                  - #             1652                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 533 amino - #acids                                                 (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                               - - Met Ala Glu Arg Ala Gln Arg Lys Ala Phe Le - #u Gln Ala Arg Asn Cys       1               5   - #                10  - #                15                - - Ile Glu Asp Arg Leu Arg Leu Glu Asp Glu As - #n Glu Lys Gln Glu Arg                   20      - #            25      - #            30                    - - Leu Leu Met Ser Leu Leu Pro Arg Asn Val Al - #a Met Glu Met Lys Glu               35          - #        40          - #        45                        - - Asp Phe Leu Lys Pro Pro Glu Arg Ile Phe Hi - #s Lys Ile Tyr Ile Gln           50              - #    55              - #    60                            - - Arg His Asp Asn Val Ser Ile Leu Phe Ala As - #p Ile Val Gly Phe Thr       65                  - #70                  - #75                  - #80         - - Gly Leu Ala Ser Gln Cys Thr Ala Gln Glu Le - #u Val Lys Leu Leu Asn                       85  - #                90  - #                95                - - Glu Leu Phe Gly Lys Phe Asp Glu Leu Ala Th - #r Glu Asn His Cys Arg                   100      - #           105      - #           110                   - - Arg Ile Lys Ile Leu Gly Asp Cys Tyr Tyr Cy - #s Val Ser Gly Leu Thr               115          - #       120          - #       125                       - - Gln Pro Lys Thr Asp His Ala His Cys Cys Va - #l Glu Met Gly Leu Asp           130              - #   135              - #   140                           - - Met Ile Asp Thr Ile Thr Ser Val Ala Glu Al - #a Thr Glu Val Asp Leu       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Asn Met Arg Val Gly Leu His Thr Gly Arg Va - #l Leu Cys Gly Val         Leu                                                                                              165  - #               170  - #               175              - - Gly Leu Arg Lys Trp Gln Tyr Asp Val Trp Se - #r Asn Asp Val Thr Leu                   180      - #           185      - #           190                   - - Ala Asn Val Met Glu Ala Ala Gly Leu Pro Gl - #y Lys Val His Ile Thr               195          - #       200          - #       205                       - - Lys Thr Thr Leu Ala Cys Leu Asn Gly Asp Ty - #r Glu Val Glu Pro Gly           210              - #   215              - #   220                           - - His Gly His Glu Arg Asn Ser Phe Leu Lys Th - #r His Asn Ile Glu Thr       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Phe Phe Ile Val Pro Ser His Arg Arg Ala Al - #a Ala Gly Gly Met         Pro                                                                                              245  - #               250  - #               255              - - Pro Ala Ala Ala Gly Gly Met Arg Gln Ser Gl - #u Tyr Tyr Cys Arg Leu                   260      - #           265      - #           270                   - - Asp Phe Leu Trp Lys Asn Lys Phe Lys Lys Gl - #u Arg Glu Glu Ile Glu               275          - #       280          - #       285                       - - Thr Met Glu Asn Leu Asn Arg Val Leu Leu Gl - #u Asn Val Leu Pro Ala           290              - #   295              - #   300                           - - His Val Ala Glu His Phe Leu Ala Arg Ser Le - #u Lys Asn Glu Glu Leu       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Tyr His Gln Ser Tyr Asp Cys Val Cys Val Me - #t Phe Ala Ser Ile         Pro                                                                                              325  - #               330  - #               335              - - Asp Phe Lys Glu Phe Tyr Thr Glu Ser Asp Va - #l Asn Lys Glu Gly Leu                   340      - #           345      - #           350                   - - Glu Cys Leu Arg Leu Leu Asn Glu Ile Ile Al - #a Asp Phe Asp Asp Leu               355          - #       360          - #       365                       - - Leu Ser Lys Pro Lys Phe Ser Gly Val Glu Ly - #s Ile Lys Thr Ile Gly           370              - #   375              - #   380                           - - Ser Thr Tyr Met Ala Ala Thr Gly Leu Ser Al - #a Ile Pro Ser Gln Glu       385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - His Ala Gln Glu Pro Glu Arg Gln Tyr Met Hi - #s Ile Gly Thr Met         Val                                                                                              405  - #               410  - #               415              - - Glu Phe Ala Tyr Ala Leu Val Gly Lys Leu As - #p Ala Ile Asn Lys His                   420      - #           425      - #           430                   - - Ser Phe Asn Asp Phe Lys Leu Arg Val Gly Il - #e Asn His Gly Pro Val               435          - #       440          - #       445                       - - Ile Ala Gly Val Ile Gly Ala Gln Lys Pro Gl - #n Tyr Asp Ile Trp Gly           450              - #   455              - #   460                           - - Asn Thr Val Asn Val Ala Ser Arg Met Asp Se - #r Thr Gly Val Leu Asp       465                 4 - #70                 4 - #75                 4 -       #80                                                                               - - Lys Ile Gln Val Thr Glu Glu Thr Ser Leu Il - #e Leu Gln Thr Leu         Gly                                                                                              485  - #               490  - #               495              - - Tyr Thr Cys Thr Cys Arg Gly Ile Ile Asn Va - #l Lys Gly Lys Gly Asp                   500      - #           505      - #           510                   - - Leu Lys Thr Tyr Phe Val Asn Thr Glu Met Se - #r Arg Ser Leu Ser Gln               515          - #       520          - #       525                       - - Ser Asn Leu Ala Ser                                                           530                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 6 amino - #acids                                                   (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                  - -    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                - - Ala Ala Ala Gly Gly Met                                                   1               5                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 14 amino - #acids                                                  (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                               - - Ala Ala Ala Gly Gly Met Pro Pro Ala Ala Al - #a Gly Gly Met               1               5   - #                10                                       - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 22 amino - #acids                                                  (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                  - -    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                - - Ala Ala Ala Gly Gly Met Pro Pro Ala Ala Al - #a Gly Gly Met Pro Pro       1               5   - #                10  - #                15                - - Ala Ala Ala Gly Gly Met                                                               20                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 29 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                               - - GGCCGCTCAC CATCACCATC ACCATTAGG         - #                  - #                 29                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 29 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                               - - AATTCCTAAT GGTGATGGTG ATGGTGAGA         - #                  - #                 29                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:24:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 24 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                               - - GATCCATCAT GAGACAGAGT GAAT          - #                  - #                     24                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:25:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 20 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                               - - ATTCACTCTG TCTCATGATC            - #                  - #                       - # 20                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:26:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 12 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                               - - GGCCGCTGGA GG              - #                  - #                       - #       12                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:27:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 12 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                               - - GATGCCTCCA GC              - #                  - #                       - #       12                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:28:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 14 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                               - - GATCTAGCTA GCTA              - #                  - #                       - #     14                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:29:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 10 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                               - - TAGCTAGCTA                - #                  - #                       - #        10                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:30:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 29 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                               - - ATGAGATCTG GATGCCAAGT TGCTCTGAG         - #                  - #                 29                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:31:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 23 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                               - - TGGAGTCATG ACACAGAGTG AAT           - #                  - #                     23                                                                     __________________________________________________________________________ 

What is claimed is:
 1. A soluble mammalian polypeptide composition having adenylyl cyclase activity, wherein said polypeptide is activated by G_(s)α.
 2. The polypeptide composition according to claim 1, wherein said polypeptide composition comprises one or more polypeptides that lack transmembrane regions.
 3. The polypeptide composition according to claim 2, wherein said polypeptide composition comprises a chimera of adenylyl cyclase C₁ and C₂ domains linked covalently.
 4. The polypeptide composition according to claim 3, wherein said chimera comprises adenylyl cyclase type I-C₁ and type II-C₂ domains, but lacks membrane-bound domains.
 5. The polypeptide composition according to claim 3, wherein said chimera comprises adenylyl cyclase type V-C₁ and type II-C₂ domains, but lacks membrane-bound domains.
 6. The polypeptide composition according to claim 4, wherein said type I-C₁ domain has a C_(1a) domain sequence from SEQ ID NO:2 and said type II-C₂ domain has a C_(2a) domain sequence from SEQ ID NO:4.
 7. The polypeptide composition according to claim 6, wherein said C_(1a) and C_(2a) domains are joined by a linker peptide.
 8. The polypeptide composition according to claim 7, wherein said linker peptide has a sequence selected from the group consisting of AAAGGM (SEQ ID NO:19), AAAGGMPPAAAGGM (SEQ ID NO:20) and AAAGGM(PPAAAGGM)₂ (SEQ ID NO:21).
 9. The polypeptide composition according to claim 7, wherein said chimera has the amino acid sequence of SEQ ID NO:18.
 10. The polypeptide composition according to claim 2, wherein said polypeptide composition forms a complex comprising two distinct polypeptides, one of which is an adenylyl cyclase C₁ domain and one of which is an adenylyl cyclase C₂ domain.
 11. The polypeptide composition according to claim 10, wherein said C₁ domain is a type I C₁ domain and said C₂ domain is a type II C₂ domain.
 12. The polypeptide composition according to claim 10, wherein said C₁ domain is a type V C₁ domain and said C₂ domain is a type II C₂ domain.
 13. The polypeptide composition according to claim 11, wherein said type I-C₁ domain has a C_(1a) domain sequence from SEQ ID NO:2 and said type II-C₂ domain has a C_(2a) domain sequence from SEQ ID NO:4.
 14. A polynucleotide encoding a soluble mammalian polypeptide having adenylyl cyclase activity, wherein said polypeptide is activated by G_(s)α.
 15. The polynucleotide according to claim 14, wherein said polynucleotide does not encode transmembrane regions.
 16. The polynucleotide according to claim 15, wherein said polynucleotide encodes a chimera of adenylyl cyclase C₁ and C₂ domains.
 17. The polynucleotide according to claim 16, wherein said polynucleotide has the nucleotide sequence of SEQ ID NO:17.
 18. A polynucleotide consisting of a coding region for an adenylyl cyclase C₁ domain.
 19. A polynucleotide consisting of a coding region for an adenylyl cyclase C₂ domain.
 20. An expression vector comprising a polynucleotide encoding a soluble mammalian polypeptide having adenylyl cyclase activity, operably linked to a promoter, wherein said polypeptide is activated by G_(s)α.
 21. An expression vector comprising a polynucleotide, wherein said polynucleotide comprises a coding region for at least one of an adenylyl cyclase C₁ or C₂ domain, operably linked to a promoter, but lacks a coding region for an adenylyl cyclase domain that is membrane bound in situ.
 22. A host cell comprising the expression vector of claim
 20. 23. The host cell of claim 22, wherein the cell is (i) a bacterial cell or (ii) a cell capable of supporting baculovirus replication.
 24. The host cell of claim 22, further comprising an expression vector comprising a polynucleotide encoding the alpha subunit of G protein, operably linked to a promoter active in said host cell.
 25. A host cell comprising the expression vector of claim
 21. 26. A host cell comprising an expression vector comprising a polynucleotide, wherein said polynucleotide comprises a coding region for at least one of an adenylyl cyclase C₁ or C₂ domain, operably linked to a promoter, but lacks a coding region for adenylyl cyclase domain that is membrane bound in situ.
 27. The host cell of claim 26, further comprising an expression vector comprising a polynucleotide encoding the alpha subunit of G protein, operably linked to a promoter active in said host cell. 